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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2158-2166.)
© 1997 American Heart Association, Inc.

Articles

HDL and ApoA Prevent Cell Death of Endothelial Cells Induced by Oxidized LDL

Isabelle Suc; Isabelle Escargueil-Blanc; Muriel Troly; Robert Salvayre; ; Anne Nègre-Salvayre

From the Department of Biochemistry, INSERM U.466, IFR Louis Bugnard, University Paul Sabatier, Toulouse, France.

Correspondence to Dr A. Nègre-Salvayre or Prof R. Salvayre, Laboratoire de Biochimie et INSERM U.466, C.H.U. Rangueil-1, Ave Jean Poulhès, 31403 Toulouse Cedex 4, France. E-mail salvayre{at}rangueil.inserm.fr

	Abstract

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Abstract We have previously demonstrated that toxic doses of mildly oxidized LDL evokes in cultured cells a delayed and sustained rise of cytosolic [Ca²⁺], eliciting in turn irreversible cell damage and leading finally to cell death. HDL and delipidated apolipoprotein (apo) A prevented effectively the toxic effect of oxidized LDL to bovine aortic endothelial cells, in a time- and dose-dependent manner. The major part of the protective effect was mimicked by purified apoA-I, whereas purified apoA-II exhibited only very low protective activity. The protective effect was independent of the paraoxonase-linked HDL activity. The protective effect of HDL is independent of the contact of HDL with oxidized LDL, as shown by preincubation of oxidized LDL with HDL or apoA. In contrast, the protective effect was dependent on the integrity of apoA and on the contact of HDL with cells, thus suggesting that HDL acts directly on cells by enhancing their resistance against oxidized LDL. Preincubation experiments show that the protective effect is dependent on the duration of the contact of cells with HDL (maximal effect observed after 12 to 16 hours' preincubation), is also dependent on protein synthesis, and is persistent for at least 48 hours after the end of the contact of HDL with cells. Finally, effective concentrations of HDL inhibit the Ca²⁺ peak, which is directly involved in the cytotoxic effect of oxidized LDL, as shown by the inhibitory effect of Ca²⁺ chelators. All together, these results suggest that HDL, mainly apoA-I, increases the resistance of endothelial cells against oxidized LDL and prevents its toxic (apoptotic) effect by blocking the pathogenic intracellular signaling (culminating in sustained Ca²⁺ rise) involved in cell death.

Key Words: lipoproteins • HDL • oxidized LDL • apoA • apoptosis • toxicity • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Low-density lipoprotein plays an important role in delivering cholesterol to animal peripheral cells.¹ In humans, among the multiple risk factors of atherosclerosis, LDL levels are highly predictive of coronary heart disease, one of the most prevalent causes of morbidity and mortality in Western countries.² LDL is thought to play an important role in atherogenesis^{1 3} after undergoing oxidative modifications.^{4 5}

LDL oxidation can be mediated by free radicals in the presence of transition metals ^{6 7 8} or cultured cells,⁹ or by UV or radiation.^{10 11 12} LDL oxidation is a progressive process leading at first to the formation of mildly oxidized LDL, which is defined by low content of lipid-peroxidation derivatives and slight apoB modifications, and later to extensively oxidized LDL which contains high levels of lipid-peroxidation products and severe apoB alterations ^{5 6 7} .

Oxidized LDL is present in atherosclerotic lesions^{13 14} and thought to be formed in vivo in the arterial wall.⁵ Oxidized LDL exhibits a wide range of biological properties potentially involved in atherogenesis.^{5 13} Oxidized LDL has been shown to be cytotoxic to cultured endothelial cells.^{15 16 17} We have recently reported that mildly oxidized LDL induces massive apoptosis of cultured human endothelial cells, followed by cell detachment and postapoptotic necrosis of floating cells.¹⁸ This toxic effect of oxidized LDL may explain the defects of endothelial cell lining observed on the surface of atherosclerotic lesions and may increase the local permeability, vascular tone, and thrombogenicity, resulting finally in local vascular dysfunction and thrombotic events.¹⁹

Disruption of Ca²⁺ homeostasis seems to play a critical role in the mechanism of toxic cell injury by triggering Ca²⁺-dependent injurious processes; for instance, activation of degrading enzymes, resulting in irreversible damage of cellular components and leading to cell death.^{20 21 22} We have recently reported that oxidized LDL induced a delayed and sustained rise of cytosolic [Ca²⁺], which induced necrosis and apoptosis on lymphoid cells.^{23 24}

HDL levels have been shown to be inversely correlated with the risk of coronary heart disease,²⁵ but the mechanisms of the protective effect of HDL are not fully understood. Besides its role in cholesterol reverse transport (from peripheral tissues to the liver),^{26 27} HDL exhibited a protective effect against the cytotoxicity of oxidized LDL by inhibiting LDL oxidation induced by cells²⁸ and inhibiting the cytotoxicity of oxidized LDL to cultured cells.¹⁶ To our knowledge, the mechanism of the protective effect of HDL at the cellular level is still unknown.

We report in the present study that (1) HDL and delipidated apoA prevented very effectively the cytotoxicity of cultured endothelial cells induced by oxidized LDL, (2) the protective effect of apoA-I was significantly higher than that of apoA-II, (3) HDL and apoA act at the cellular level by increasing the resistance of endothelial cells against the cytotoxicity of oxidized LDL during a relatively long time (protective activity remaining for several days), and (4) HDL inhibits the pathogenetic intracellular signaling triggered by oxidized LDL that induces the sustained Ca²⁺ rise leading to cell death.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Chemicals
[³H]Cholesteryl oleyl ether (38 Ci/mmol) was from Amersham; EDTA, EGTA, MTT, TNBS, BSA, apoA-I, and apoA-II were from Sigma Chemical Company; thiobarbituric acid and 1,1,3,3-tetraethoxypropane were from Fluka; quin 2-AM and fura 2-AM were from Molecular Probes; DMEM, phenol red–free–DMEM, RPMI-1640, fetal calf serum, penicillin, and streptomycin were from GIBCO; and Ultroser G was from IBF. Multiwell culture plates were from Nunc (distributed by Polylabo-Block), Hydragel was from Sebia, and the other reagents and chemicals were obtained from Merck or Prolabo.

Cell Culture
BAECs GM7372A were obtained from the NIG Human Mutant Cell Repository (Camden, NJ); the human umbilical vein endothelial cell line (CRL-1998) was obtained from American Type Culture Collection (Rockville, MD); and the (nonimmortalized) BAECs were obtained according to Gospodarowicz et al,²⁹ as previously used.³⁰ Cells were grown in RPMI-1640 (CRL-1998) and DMEM (GM7372A and BAECs were used between the 5th and 10th passages). All passages were made by using a splitting ratio of 1:4. Under standard conditions, endothelial cells (0.4x10⁶ per milliliter) were seeded in 6- or 24-multiwell plates or in falcons (Nunc) when required, grown in their respective medium with Glutamax supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The culture medium of BAECs was supplemented with 1 ng/mL basic fibroblast growth factor. Cells were incubated in a humidified incubator (Heraeus; 5% CO₂, 37°C). One day before LDL incorporation, this medium was replaced by a serum-free medium. In this study we used mainly the bovine GM7372A cells, because this cell line is more stable and less fragile (thus less susceptible to interfering cell death) than the nonimmortalized BAECs and does not induce LDL oxidation, in contrast to the human CRL-1998 cell line.

Isolation, Labeling of Lipoproteins, and Uptake Determination
Pooled sera obtained from healthy donors were heat treated (1 hour at 56°C) and LDL was isolated by sequential ultracentrifugation (Beckman L8-70 ultracentrifuge) according to Havel et al³¹ and extensively dialyzed against 0.15 mol/L NaCl containing 0.3 mmol/L EDTA. The REM was evaluated by electrophoresis on Hydragel. LDL was sterilized by filtration (0.2-µm Millipore membrane) and stored at 4°C under nitrogen (up to 2 weeks). ApoB and apoA-I concentrations were determined by immunonephelometry (Behring system).

LDL was labeled with [³H]cholesteryl oleyl ether (10⁵ dpm per milligram apoB) according to Roberts et al,³² isolated again by ultracentrifugation, dialyzed and sterilized on a 0.2-µm Millipore membrane, and kept under nitrogen at +4°C until use, under the previously described conditions.²⁴ Radiolabeled (oxidized) LDL (200 µg apoB or apoA-I per milliliter) was added to the culture medium for 12 hours (in the presence or absence of nonlabeled LDL or HDL, as indicated in the related figures), and cells were carefully washed twice in phosphate buffered saline, harvested, and homogenized by sonication in 1 mL of distilled water. An aliquot was used for determining the cell-associated radioactivity (liquid scintillation counter, Packard model Tricarb 4530).

Delipidated apoA was obtained from purified HDL according to the procedure of Osborne.³³ HDL or delipidated apoA was iodinated according to the procedure of Weech et al,³⁴ under the previously used conditions.³⁵ Briefly, 2 mg apoA in 0.2 mol/L phosphate buffer, pH 7.5, was mixed with 0.5 mCi ¹²⁵I and 2.5 µg chloramine T (three times every 30 seconds). The reaction was stopped by adding 250 µg tyrosine, and the lipoproteins were extensively dialyzed as indicated above. Ninety-five percent of the radioactivity was TCA precipitable. The ¹²⁵I radioactivity was 45 000 dpm per microgram apoA (determined by a gamma counter, Packard model Minaxi 5000). Cells were incubated for 14 hours in serum-free RPMI-1640 containing 200 µg apoA per milliliter (and 890,000 dpm/mL). Then cells were extensively washed, four times with RPMI-1640 containing 10% fetal calf serum and three times with RPMI-1640 (without serum). After washing, cells were incubated for the indicated time at 37°C in serum-free RPMI-1640. At each time, cells were washed as indicated above and incubated with trypsin plus EDTA for 10 minutes at 37°C. Cells were pelleted by centrifugation (800g for 5 minutes), and the ¹²⁵I radioactivity of the supernatant (trypsin releasable) and the cell pellet (trypsin resistant) was determined. At the indicated time, the culture medium was collected, TCA (final concentration 150 mg/mL) was added successively and allowed to react for 30 minutes at 0°C, and the mixture was centrifuged (3000g for 10 minutes). The TCA-soluble (supernatant) and TCA-insoluble (pellet solubilized in 0.1N NaOH containing 20 mg/mL SDS for 4 hours at 37°C) portions were counted for ¹²⁵I.

LDL Oxidation
Mildly oxidized LDL was obtained by UV treatment of 2 mg apoB per milliliter of LDL (in 150 mmol/L NaCl containing 100 µmol/L EDTA), irradiated by UV-C (254 nm, 500 µW/cm², Osram UV lamp, for 2 hours) in the presence of 2 µmol/L CuSO₄, as previously used.²⁴

Cell-mediated oxidation was performed by incubating LDL (0.2 mg apoB per milliliter) for 16 hours with human preconfluent endothelial cells (CRL-1998) in serum-free phenol red–free–RPMI-1640 medium, under the previously used conditions.¹⁸ At the end of the oxidation time, the cell-culture medium containing cell-oxidized LDL (comparable to LDL-free culture medium of lymphoblastoid cells used as control) was transferred to bovine endothelial cells (GM7372A). Under the experimental conditions used here, the bovine endothelial cell line did not significantly oxidize the LDL added to the culture medium (in contrast to the human endothelial cell line).

LDL oxidation was evaluated by monitoring the level of TBARS formation according to Yagi³⁶ and the REM on Hydragel and by determining the TNBS-reactive amino groups determined according to Steinbrecher.³⁷

Usually "native" (ie, nonoxidized) LDL contained between 0.1 and 0.3 nmol TBARS per milligram apoB. Under the standard conditions used here, mildly (UV) oxidized LDL was characterized by relatively low levels of TBARS content (4 to 6 nmol per milligram apoB) without major alteration of apoB (REM 1.2±0.1 and TNBS 98±2%). Cell-oxidized LDL contained relatively low levels of TBARS (3.5±0.4 nmol per milligram apoB) without major alteration of apoB (REM 1.15±0.1 and TNBS 96±3%).

Determination of Cytosolic [Ca²⁺]
[Ca²⁺]_i was determined according to the methods Arslan et al³⁸ and Grynkiewicz et al,³⁹ by using the permeant Ca²⁺ probes quin 2-AM or fura 2-AM, which are hydrolyzed by intracellular carboxylesterases to liberate quin 2 or fura 2. Briefly, cells were incubated for 15 minutes at 37°C in DMEM buffered with 20 mmol/L HEPES and containing 0.5% BSA and quin 2-AM (20 µmol/L) or fura 2-AM (5 µmol/L). After dilution and incubation in DMEM for 45 minutes, cells were washed twice in phosphate buffered saline and their fluorescence was recorded. With quin 2 (excitation 340 nm, emission 395 nm), the calibration was done in very low and high Ca²⁺ to calculate the absolute value of [Ca²⁺]_i, as described by Arslan et al.³⁸ With fura 2, [Ca²⁺]_i determination was performed at the dual excitation wavelength of 340 and 380 nm and emission at 510 nm. [Ca²⁺]_i was calculated by the ratio method of Grynkiewicz et al.³⁹ Both fluorescent Ca²⁺ probes gave quite similar results; therefore, to avoid redundant data, we report only results obtained with one probe.

Cytotoxicity Determination
For determining the cytotoxicity (and the cytoprotective effect), oxidized LDL and/or HDL (or apoA) was added to the culture medium of BAECs plated in 96- or 24-microwell tissue-culture dishes. Under standard conditions, the whole cytotoxicity was determined after 48 hours' incubation, by using two tests concomitantly (to exclude artifacts): (1) the MTT test according to Price and McMillan,⁴⁰ with the following modifications: cells were incubated for 0.5 hours in 50 µL phenol red–free RPMI-1640 containing 2 mmol/L MTT, then 200 µL DMSO was added and the absorbance at 530 nm was read with a Titertek Labsystems spectrophotometer and (2) the CFDA test of McGinnes et al.⁴¹ Because the two tests gave similar results, we report the data of only one to avoid redundant data.

Paraoxonase Activity Determination
Paraoxonase activity was determined by using paraoxon as substrate and measuring spectrophotometrically the p-nitrophenol formation at 410 nm, according to Watson et al.⁴²

Proteins were determined by the method of Lowry et al.⁴³ Results are expressed as mean±SEM, and the statistical significance was estimated by Student's t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
HDL and ApoA Prevent Endothelial Cell Death Induced by Toxic Doses of Oxidized LDL
The mildly oxidized LDL in our study was prepared by using UV+copper/EDTA oxidation, because this method allows control of the oxidation level and easier preparation of mildly oxidized LDL than does copper-promoted oxidation and also allows the exclusion of biologically active compounds (such as cytokines or growth factors) that are present in cell-mediated oxidized LDL preparations and potentially interfere with endothelial cells. Under the experimental conditions used, mildly (UV+copper) oxidized LDL contained relatively low levels of TBARS (4.4±0.3 nmol per milligram apoB) and slight alterations of REM (1.2±0.1 in comparison with native LDL) and TNBS reactivity (92±2% of the native LDL). We observed a large interindividual variability in the susceptibility of LDL to oxidation and in the subsequent toxic effect of oxidized LDL. To decrease this variability, we used serum pooled from a large number of donors (100 to 200).

Oxidized LDL exhibited a time- and dose-dependent cytotoxicity to GM7372A endothelial cells, whereas native (nonoxidized) LDL was not or only poorly cytotoxic over the range of concentrations used here (up to 200 µg apoB per milliliter; Fig 1A and 1B). HDL (200 µg apoA per milliliter) added to the culture medium simultaneously with oxidized LDL prevented the toxic effect of oxidized LDL (Fig 1A). This protective effect was dose dependent (Fig 1C). We observed, however, that the protective effect was dependent on the relative concentration of oxidized LDL and HDL, ie, the protective effect of a fixed concentration of HDL was overwhelmed when oxidized LDL concentration increased (Fig 1B). Under the same experimental conditions, delipidated apoA exhibited also a significant protective effect (although lower than that of HDL) (Fig 1B and 1C), suggesting that the major part of the cytoprotective effect of HDL is associated with the apoprotein fraction. Moreover, experiments with purified apoproteins showed that the major part of the cytoprotective effect may be attributed to apoA-I, whereas the protective activity of apoA-II was relatively low (Fig 1C).

View larger version (23K): [in this window] [in a new window]   Figure 1. Protection of HDL against the cytotoxic effect of oxidized LDL on cultured endothelial cells. A, Time course of toxicity evaluated in the presence of native LDL (200 µg apoB per milliliter, ), oxidized LDL (200 µg apoB per milliliter, ), or oxidized LDL (200 µg apoB per milliliter) plus HDL (200 µg apoB per milliliter and 200 µg/mL apoA, ) added simultaneously at time 0. B, Dose dependence of the cytotoxic effect of increasing concentrations of oxidized LDL used alone () or with a fixed dose (200 µg apoA per milliliter) of HDL () or delipidated apoA (), added simultaneously to the culture medium. C, Protective effect of increasing concentrations of HDL (), apoA mixture, purified apoA-I, or purified apoA-II () against the toxicity of a fixed dose of oxidized LDL (200 µg apoB per milliliter). Toxicity was evaluated by the MTT test, expressed as percent of the control (ie, cells grown under the same conditions but in the absence of any lipoprotein). Mean±SEM of three experiments.

To examine the relevance of our experimental model system, we have compared the protective effect of HDL observed in this system (oxidation by UV+copper) with that observed with LDL oxidized by cells, a more (patho) physiological system. A similar toxic effect of oxidized LDL and a similar protection of HDL was observed in the two experimental systems (Table 1). Because preparations of cell-oxidized LDL contain molecules secreted by cells during LDL oxidation and potentially interacting with endothelial cells, we prefer to use UV+copper–oxidized LDL (which is not contaminated by such biological compounds).

View this table: [in this window] [in a new window]   Table 1. Comparison of the Protective Effect of HDL Against the Cytotoxic Effects of Mildly (UV) Oxidized or Cell-Oxidized LDL to the Immortalized BAEC Line GM7372A

In the same way, to verify that the conclusions about the toxic effect of oxidized LDL and protection by HDL were not restricted to the GM7372A cell line), we used a human immortalized endothelial cell line (CRL-1998) and nonimmortalized BAECs for comparison. The data obtained with CRL-1998 cells were quite similar to those obtained with GM7372A. Nonimmortalized BAECs were relatively more susceptible to the toxic effect of oxidized LDL, but HDL was also able to reduce the toxicity of oxidized LDL (Table 2).

View this table: [in this window] [in a new window]   Table 2. Comparison of the Protective Effect of HDL Against the Cytotoxic Effects of Mildly Oxidized LDL to Three Types of Endothelial Cells: CRL-1998, GM7372A, and BAECs

In the above-reported experiments (coincubation of cells with HDL and oxidized LDL), the protective effect of HDL might result from (1) the interaction of HDL with oxidized LDL, leading to reduced amounts of cytotoxic compounds in oxidized LDL²⁸ ; (2) the inhibition of the cellular uptake of oxidized LDL, thereby reducing its cytotoxicity, which has been shown to be related to the amount of LDL taken up by cells⁴⁴ ; and (3) the interaction of HDL with cells, leading to increased cellular resistance against the cytotoxic effect of oxidized LDL. These hypotheses have been tested in experiments reported below.

Preincubation of Oxidized LDL With HDL Does Not Reduce Their Cytotoxic Effect
Under the experimental conditions used here, when oxidized LDL was preincubated with HDL (for 14 or 24 hours) or with apoA (for 6 hours) and separated again, the cytotoxic effect of the oxidized LDL (preincubated with HDL or apoA) was not significantly different from that of the control (ie, oxidized LDL preincubated under the same conditions but without HDL or apoA; Table 3). Therefore, it may be concluded that the protective effect of HDL and apoA cannot be attributed to the interaction of oxidized LDL with HDL (nor to the inactivation or extraction of toxic compounds by HDL or apoA).

View this table: [in this window] [in a new window]   Table 3. Preincubation of Oxidized LDL With HDL or ApoA

HDL and ApoA Do Not Inhibit the Uptake of Oxidized LDL by Endothelial Cells
The rate of uptake of mildly oxidized LDL is a critical parameter for the cytotoxic effect to cultured cells, since we have previously shown that the cytotoxicity of oxidized LDL is dependent on the amount of oxidized LDL internalized by cells.⁴⁴ Therefore, we investigated whether HDL could reduce the uptake of oxidized LDL by endothelial cells and reduce subsequently their cytotoxic effect. As shown in Table 4, the concentration of HDL and apoA used here (200 µg apoA per milliliter) did not alter significantly the uptake of oxidized LDL (radiolabeled with [³H]cholesteryl oleyl ether) by endothelial cells. In contrast, native (unlabeled) LDL inhibited competitively the uptake of labeled mildly oxidized LDL, thus confirming that mildly oxidized LDL is mainly taken through the apoB/E pathway (because apoB is not or only slightly modified).⁴⁴ These data suggest that the cytoprotective effect of HDL does not result from a reduced cellular influx of toxic molecules contained in oxidized LDL.

View this table: [in this window] [in a new window]   Table 4. Effect of HDL or ApoA on the Uptake of Oxidized LDL

Preincubation of Endothelial Cells with HDL Prevents the Cytotoxic Effect of Oxidized LDL
Since under the conditions we used, the protective effect of HDL and apoA cannot be attributed to an extraction of toxic compounds from oxidized LDL nor to an inhibition of oxidized LDL uptake by cells, we investigated the role of HDL-cell interaction in the protective effect of HDL.

Preincubation of endothelial cells with HDL or apoA before incubation with toxic doses of oxidized LDL prevented the toxic effect of oxidized LDL to endothelial cells, thus suggesting that HDL enhances the resistance of cells against oxidized LDL.

When cells were preincubated with HDL for variable periods of time, the cytoprotective effect was not immediate. Preincubation of cells with HDL for less than 5 hours did not confer a significant protection, whereas preincubation for 10 hours and longer induced a progressively increasing protective effect, which was near maximal after 20 hours' preincubation (Fig 2A). This maximal protection obtained after 20 hours' preincubation needs a continuous contact between HDL and cells, since 10 hours' contact (between HDL and cells) followed by a 10-hour interval without HDL did not induce any increase in the protection in comparison with 10 hours' contact without interval (Fig 2B). This finding suggests that the progressive increase of cellular defenses may result from a continuous stimulation leading to a maximal cellular resistance or/and from a progressive accumulation of apoA in cells.

View larger version (24K): [in this window] [in a new window]   Figure 2. Protective effect of HDL against the toxic effect of oxidized LDL, dependent on the time of contact between HDL and cells. A, Endothelial cells were preincubated for variable times (0 to 20 hours) in serum-free medium containing a fixed dose of HDL or apoA (200 µg apoA per milliliter), washed once with fresh medium, and incubated in the presence of oxidized LDL (200 µg apoB per milliliter) for an additional 48 hours. The cytotoxic effect was evaluated by the MTT test at the end of the 48-hour incubation time. B, Before incubation with oxidized LDL (200 µg apoB per milliliter for 48 hours), cells were preincubated without HDL (a) or with HDL (200 µg apoA per milliliter) for 10 or 20 hours (b and c, respectively) or for 10 hours with HDL followed by 10 hours without HDL (d). Mean±SEM of three experiments.

Cell-associated HDL did not result from (nonspecific) fluid phase pinocytosis but rather from a specific binding of HDL, since, as shown by competition experiments, cell association of ¹²⁵I-HDL was inhibited by unlabeled HDL but not by LDL (Table 5). Conversely, the protective effect of HDL was dependent on the integrity of apoA, since proteolysis of apoA (by trypsinization) inhibited the protective effect (Table 6).

View this table: [in this window] [in a new window]   Table 5. Effect of Unlabeled HDL or LDL on the Level of Cell-Associated 125I-HDL

View this table: [in this window] [in a new window]   Table 6. Effect of Trypsinization on the Protective Effect of HDL or ApoA Against the Toxic Effect of Oxidized LDL to Endothelial Cells

When cells were preincubated with HDL for 24 hours, grown in an HDL-free medium for a variable interval of time, and finally incubated for 48 hours in an HDL-free medium containing oxidized LDL, the cytoprotective effect persisted for more than 36 and 24 hours with HDL and apoA, respectively (Fig 3). To examine whether this protective effect was related to a persistent association of apoA with endothelial cells, we performed pulse-chase experiments with ¹²⁵I-apoA. As reported in Fig 4, about 50% of the cell-associated ¹²⁵I-apoA was rapidly (in 5 to 7 hours) released in the culture medium (as TCA-soluble ¹²⁵I-labeled material). The 50% remaining ¹²⁵I-labeled fraction was cell associated and was, in major part, trypsin resistant.

View larger version (23K): [in this window] [in a new window]   Figure 3. Protective effect of HDL against the toxic effect of oxidized LDL. This effect is persistent after the contact of HDL with cells. Endothelial cells were preincubated for 24 hours in serum-free medium containing a fixed dose of HDL (200 or 400 µg apoA per milliliter) or apoA (200 µg apoA per milliliter), washed once with fresh medium, and after variable intervals of time (0 to 60 hours) incubated in the presence of oxidized LDL (200 µg apoB per milliliter) for an 48 hours. At the end of the 48-hour incubation time, the cytotoxic effect was evaluated by the MTT test. Mean±SEM of three experiments.

View larger version (24K): [in this window] [in a new window]   Figure 4. Association of 125I-apoA to and release from endothelial cells. Cells were pulsed with 125I-apoA (200 µg apoA per milliliter containing 890 000 dpm/mL) for 14 hours at 37°C in serum-free medium. At the end of the pulse, cells were thoroughly washed, as described in "Methods," and "chased" for variable times (0 to 48 hours) in the (HDL- and serum-free) culture medium. After washing, cells were harvested by trypsinization (trypsin/EDTA for 10 minutes at 37°C). Cells were pelleted by centrifugation (800g for 5 minutes), and the 125I radioactivity of the supernatant (trypsin releasable; ) and cell pellet (trypsin resistant; ) was determined. The culture medium was collected, TCA (final concentration 150 mg/mL) added and allowed to react for 30 minutes at 0°C, centrifuged (3000g for 10 minutes), and the radioactivity of the TCA-soluble () and TCA-insoluble () fractions was counted for 125I. Mean±SEM of three experiments.

The comparison of experiments of Figs 3 and 4 showed that under conditions employing a time interval of 12 hours between apoA preincubation and incubation with oxidized LDL, an effective protection was observed (Fig 3), whereas the level of the cell-associated apoA decreased only very slightly during the period between 12 and 48 hours (Fig 4) (which corresponds to the incubation with oxidized LDL in Fig 3). Since, under serum-free culture conditions used here and during this 12- to 48-hour period, cells released only minor amounts of apoA, it is very unlikely that the protective effect resulted from any direct "extraction" (similar to the reverse transport of cholesterol) by HDL of the cytotoxic molecules brought into the cells by oxidized LDL.

Protective Effect of HDL Is Not Correlated to Paraoxonase Activity
The values of paraoxonase activity were 163±12 nmol · min^{-1 ·}mL^-1 on the pools of serum used for lipoprotein isolation. On three batches of HDL (H1, H2 and H3), prepared under our standard conditions (dialysis against phosphate buffered saline containing 0.3 mmol/L EDTA) and kept at 4°C for 0, 2, or 4 weeks, paraoxonase activities were 1.6±0.2, 0.8±0.1, and 0.2±0.1 nmol · min^-1 · mg^-1 apoA, respectively. The three batches of HDL exhibited a similar protective activity against the toxicity of oxidized LDL: In the presence of oxidized LDL and HDL (200 µg apoB and apoA per milliliter), MTT values were 96±5%, 95±6%, and 93±5% of the control without oxidized LDL (MTT value of the control containing 200 µg apoB per milliliter oxidized LDL but no HDL was 27±4%). Moreover, in delipidated apoA batches, we detected no significant paraoxonase activity. Therefore, it may be concluded that under the experimental conditions used here, no correlation was found between paraoxonase activity and the protective activity of HDL.

Protective Effect of HDL Involves the Synthesis of Cell Proteins
When cells were incubated with oxidized LDL and HDL in the presence of 10 µmol/L cycloheximide, the protective effect of HDL and apoA was reduced (Table 7). This inhibitory effect was even more pronounced when cells were preincubated with HDL and cycloheximide. As cycloheximide was not toxic per se and did not enhance the toxicity of oxidized LDL, it is suggested from preincubation data that HDL may induce the synthesis (inhibited by cycloheximide) of proteins involved in the resistance of cells against oxidized LDL.

View this table: [in this window] [in a new window]   Table 7. Effect of Cycloheximide on the Protective Effect of HDL or ApoA Against the Cytotoxicity of Oxidized LDL to Endothelial Cells

HDL Blocks the [Ca²⁺]_i Peak Induced by Oxidized LDL and Involved Apoptosis
Toxic doses of mildly oxidized LDL have been shown to induce a Ca²⁺ influx and a delayed and sustained rise of [Ca²⁺]_i,^{23 24} which is known to be involved in the genesis of irreversible cell damage and cell death.^{21 22} In the presence of effective concentrations of HDL and apoA, this delayed and sustained [Ca²⁺]_i rise was blocked, as shown by the fluorometric determination of [Ca²⁺]_i levels on the whole cell population (Fig 5) (quite similar results were obtained by using the Ca²⁺ probes fura 2-AM and quin 2-AM). In our experimental model system, the sustained [Ca²⁺]_i rise seems to be directly involved in apoptosis induced by oxidized LDL, since an effective concentration of EGTA (0.6 mmol/L), added just before the beginning of the [Ca²⁺]_i rise blocked simultaneously the [Ca²⁺]_i rise and the apoptotic effect (Fig 5). Therefore, it is suggested that the protective effect of HDL or apoA results from their ability to inhibit the [Ca²⁺]_i rise induced by oxidized LDL.

View larger version (29K): [in this window] [in a new window]   Figure 5. Inhibitory effect of HDL on the [Ca2+]i peak evoked by oxidized LDL (200 µg apoB per milliliter). A, Time course of [Ca2+]i, determined fluorometrically on the whole cell population by using fura 2-AM. Cells were grown in six-multiwell plates. At time 0, oxidized LDL (200 µg apoB per milliliter; ) or oxidized LDL (200 µg apoB per milliliter) plus HDL (200 µg apoA per milliliter; ) was added to the culture medium. Each hour, one well was loaded with fura 2-AM (2 µmol/L) and used for [Ca2+]i determinations, as indicated in "Methods". B, Determination of [Ca2+]i at the time of the peak (12 hours) and cytotoxicity (MTT test) at 48 hours on cells grown in the absence of any additive (0/0), or with oxidized LDL (L/0), oxidized LDL (200 µg apoB per milliliter) and EGTA (0.5 mmol/L; L/E), or oxidized LDL (200 µg apoB per milliliter) and HDL (200 µg apoA per milliliter). Mean±SEM of three experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Oxidized LDL has been shown to be cytotoxic^{15 16 17} and induce apoptosis¹⁸ of cultured endothelial cells. Cell death triggered by toxic doses of oxidized LDL is mediated by the intense and sustained [Ca²⁺]_i peak subsequent to a Ca²⁺ influx, as suggested by the concomitant inhibition of the [Ca²⁺]_i peak and cytotoxicity when extracellular Ca²⁺ was chelated by EGTA and when the Ca²⁺ entry was blocked by nifedipine, a Ca²⁺ channel blocker.^{24 45}

Such endothelial injury may play a role in atherogenesis, as well as in thrombotic events.^{4 19} Although oxidized LDL (potentially cytotoxic) is present in atherosclerotic areas,^{13 14} focal defects in the endothelial surface in these areas seem to be relatively rare or transient events.¹⁹ This observation suggests the existence of powerful defense systems minimizing the toxic effects of oxidized LDL and/or of active reendothelializing repair processes.

HDL is a well-known antiatherogenic factor. Various mechanisms may be involved in this antiatherogenic effect, ie, reverse cholesterol transport,^{26 27} reduction of LDL oxidation,²⁸ and protection of the endothelium against the toxic effect of oxidized LDL.²⁵ But to our knowledge, the mechanism of this latter protective effect is not known.

The data reported here showed that HDL inhibits death of endothelial cells induced by oxidized LDL. Both HDL and delipidated apoA exhibited a protective effect. Since delipidated apoA was less effective than intact HDL (20% to 25% of activity lost during delipidation), the possibility that lipid-soluble compounds of HDL may be partly involved in the protective effect, eg, -tocopherol, which is known to be able to block the [Ca²⁺]_i peak and prevent the toxicity of oxidized LDL,⁴⁶ or that structural modifications of apoA may occur during delipidation and reduce the efficacy of apoA cannot be excluded.⁴⁷ The protective activity of the apoA mixture was mainly due to apoA-I, whereas apoA-II exhibited only a poor protective activity.

Beside the above-mentioned antiatherogenic effects of HDL, our data give some new insight into the mechanisms involved in the protective effect of HDL. Under the experimental conditions used here, the cytoprotective effect of HDL against the toxicity of oxidized LDL was independent of the ability of HDL to inhibit LDL oxidation and probably independent of the extraction of toxic compounds by HDL, since, in preincubation experiments with an interval of time (Figs 3 and 4), the protection is near maximal, in spite of the low amount of apoA released by cells. The protective effect reported here is therefore different from the protective effect of HDL against endotoxin, which is attributed to the binding and neutralization of endotoxin on the surface of HDL.⁴⁸ It seems also to be different from the inhibitory effect of apoA-I on neutrophil activation, since intact HDL has been shown to be inactive in this case.⁴⁹

The protective effect of HDL against the toxic effect of oxidized LDL was independent of the paraoxonase activity (under the experimental conditions used here), in contrast to the inhibitory effect of HDL on monocyte–endothelial cell interactions induced by oxidized LDL.⁴²

The protective effect of HDL was dependent on apoA integrity (inhibition by trypsin treatment). As suggested by pulse-chase experiments, HDL acts at the cellular level by enhancing progressively the cellular resistance against the toxicity of oxidized LDL. This resistance is dependent on protein synthesis and is persistent for at least 48 hours under the experimental conditions used here.

Toxic doses of oxidized LDL induce an intracellular signaling (largely unknown), culminating in the intense and sustained [Ca²⁺]_i rise, which is directly involved in the toxic and apoptotic effect.^{18 23 24} It was therefore postulated that HDL may induce a cellular response able to inhibit this pathogenetic signal transduction. To date, the mechanism and identity of cellular proteins that enhance cellular resistance are largely unknown, but our data (demonstrating block of the oxidized LDL-induced [Ca²⁺]_i peak by HDL) support the hypothesis that HDL blocks the pathogenic intracellular signaling triggered by oxidized LDL and the subsequent toxicity.

From a pathophysiological point of view, it may be speculated that the toxic (apoptotic) effect of oxidized LDL may be involved in the formation of focal defects of the endothelial cell lining observed in atherosclerotic areas and may therefore be involved in initiation and progression of arteriosclerosis and thrombosis.^{5 19} The results reported in the present study suggest that HDL is able to increase the cellular resistance of endothelial cells against the cytotoxicity of oxidized LDL by blocking the intracellular transduction of cytotoxic signals generated by oxidized LDL. Protection of the endothelial cell lining integrity may be one of the important functions of HDL in preventing atherogenesis.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BAEC = bovine aortic endothelial cell DMEM = Dulbecco's modified Eagle's medium REM = relative electrophoretic mobility TBARS = thiobarbituric acid–reactive substance TCA = trichloroacetic acid

	Acknowledgments

 
This work was supported by INSERM, Ministère de l'Enseignement Supérieur et de la Recherche, Conseil Régional Midi-Pyrénées, Fondation pour la Recherche Médicale, and from European Communities (PL 931790). I. Suc and I. Escargueil-Blanc were fellows of the MESR and "Ligue contre le Cancer," respectively. The authors wish to thank C. Mora for lipoprotein preparation, J.P. Estève for apoA labeling, and J. Dumoulin for technical assistance.

Received July 26, 1996; accepted March 7, 1997.

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