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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1575-1582.)
© 1997 American Heart Association, Inc.

Articles

Mitochondrial Function Is Involved in LDL Oxidation Mediated by Human Cultured Endothelial Cells

Laurence Mabile; Olivier Meilhac; Isabelle Escargueil-Blanc; Muriel Troly; Marie-Thérèse Pieraggi; Robert Salvayre; ; Anne Nègre-Salvayre

From the Biochemistry Laboratory (INSERM CJF-9206), Institut Louis Bugnard, CHU Rangueil, Toulouse, France.

Correspondence to Dr A. Nègre-Salvayre or Pr R. Salvayre, Laboratoire de Biochimie Maladies Métaboliques, CHU Rangueil, Ave Jean Poulhès, 31054 Toulouse Cedex, France.

	Abstract

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Abstract Human endothelial cells (ECs) grown under standard conditions are able to generate a basal level of oxygen free radicals and induce progressive oxidation of LDLs. Inhibition of cell-mediated LDL oxidation by superoxide dismutase, EDTA, or desferrioxamine implicates a role for superoxide anion and/or transition metals in this process. The potential role of the mitochondrion was investigated by inducing mitochondrial deenergization by selective photosensitization or the addition of inhibitors of the mitochondrial respiratory chain. Mitochondria of human cultured ECs were selectively damaged by photosensitization of cells labeled with the mitochondrion-selective fluorescent dye 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide under conditions that induced only low levels of toxicity during the time of the experiment. Photosensitized ECs exhibited severe mitochondrial dysfunction, as suggested by the defect in mitochondrial uptake of the mitochondrion-selective fluorescent dyes [rhodamine 123 and 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide] and morphological alterations as shown by transmission electron microscopy. In mitochondria-photosensitized cells, superoxide anion generation was strongly decreased, as was LDL oxidation and the subsequent cytotoxicity. When ECs were incubated with the mitochondrial respiratory-chain inhibitors antimycin A or rotenone or with the carbonylcyanide-m-chlorophenylhydrazone uncoupler rhodamine 123, uptake and subcellular distribution were altered, and concomitantly superoxide anion production and LDL oxidation were strongly decreased. In conclusion, these data suggest that mitochondrial function is required, directly or indirectly, for the production of superoxide anion and the subsequent LDL oxidation by human vascular ECs.

Key Words: LDL • oxidation • superoxide anion • mitochondria • human endothelial cells • atherosclerosis

	Introduction

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Low density lipoproteins play a major physiological role in delivering cholesterol and other lipids to peripheral cells.¹ LDLs are also involved in atherogenesis, as has been suggested by experimental, epidemiological, and clinical studies.^{1 2 3 4} OxLDLs also are thought to play a central role in atherogenesis,^{5 6 7 8 9 10} are present in atherosclerotic lesions,^{11 12 13} and exhibit a wide range of biological properties potentially involved in atherogenesis (see reviews in References 7 and 9^{7 9} ). OxLDLs are chemotactic for circulating monocytes and induce the formation of macrophage foam cells, which are characteristic of early atherosclerotic lesions.⁷ Furthermore, OxLDLs are cytotoxic, induce gene expression, are immunogenic, and alter coagulation pathways and arterial vasomotor properties.^{7 9}

LDL oxidation can be mediated by cultured cells from the vascular wall or by those present in atherosclerotic lesions (ECs, smooth muscle cells, macrophages, and lymphocytes).^{14 15 16 17} LDL oxidation is promoted by cell-derived free radicals and reactive oxygen species^{15 18 19} although the role of superoxide anion, O₂^-, has been the subject of some debate,²⁰ transition metals such as iron or copper,^{21 22} thiol compounds,²³ H₂O₂/myeloperoxidase/HClO,²⁴ and tyrosyl radicals.²⁵

In ECs, two independent mechanisms seem to be involved in LDL oxidation, the first being mediated by O₂^- and inhibited by SOD,^{15 21} and the second being (possibly and controversially) mediated by lipoxygenases.^{26 27} O₂^- and H₂O₂ may be produced in several subcellular compartments (reviewed in References 28 and 29^{28 29} ). Among the potential cellular sources of O₂^-, the mitochondrial electron transport chain is probably one of the most important pathways.^{25 28 30} O₂^- seems to be generated mainly at the NADH-Q segment and the QH2-cytochrome c segment (ie, cytochrome bcl segment) of the mitochondrial respiratory chain.²⁶ O₂^- may be dismuted to H₂O₂ in situ by mitochondrial MnSOD.^{31 32} In turn, H₂O₂ may be metabolized to water by glutathione peroxidase and catalases.^{32 33}

To study the hypothetical role of mitochondrial function in LDL oxidation by human ECs, we investigated the effect of mitochondrial deenergization (ie, mitochondria damaged by selective photosensitization under nontoxic conditions or alternatively deenergized by specific inhibitors of the respiratory chain and the uncoupler CCCP) on O₂^- production and LDL oxidation. The data reported herein strongly suggest that mitochondrial function is required for basal production of O₂^- and LDL oxidation by ECs.

	Methods

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Chemicals
RPMI-1640 with Glutamax, phenol red–free RPMI-1640, penicillin, streptomycin, and FCS were purchased from GIBCO; horse heart cytochrome c, bovine erythrocyte SOD (or superoxide:superoxide oxidoreductase, EC 1.15.1.1), xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.1.3.22) from buttermilk, trypan blue dye, TBA, antimycin A, rotenone, CCCP, L-NAME, L-NMMA, bovine liver catalase (H₂O₂:H₂O₂ oxidoreductase, EC 1.11.1.6; C9322, C30, and C40, as batches 1, 2, and 3, respectively) were from Sigma; CFDA, rhodamine 123, and DASMPI were from Molecular Probes; and the other reagents and chemicals were from Sigma, Merck, or Prolabo. Purified catalase was prepared according to the method of Steinbrecher.¹⁹

Cell Culture
The human EC line CRL-1998 was obtained from the American Type Culture Collection (Rockville, Md). Under standard conditions, the cells (4x10⁵ cells/mL) were seeded in six-multiwell plates or in falcons (Nunc); grown in RPMI-1640 medium with Glutamax supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin; and incubated in a humidified incubator (Heraeus); (5% CO₂, 37°C). Human skin fibroblasts (Chau) from normal subjects were established in our laboratory and grown under conditions similar to those for ECs as previously reported.³⁴

LDL Isolation and Oxidation
LDLs were isolated from pooled human fresh sera by ultracentrifugation according to Poumay and Ronveaux-Dupal,³⁵ dialyzed against 150 mmol/L NaCl containing 0.3 mmol/L EDTA, sterilized by filtration (0.2-µm Millipore filters), and kept at 4°C under N₂ until use (up to 3 weeks). The REM of LDL particles was controlled by electrophoresis on Hydragel (Sebia). Under the conditions used, auto-oxidation of LDL was very slow (in contrast, LDLs stored in EDTA-free solution were much more susceptible to auto-oxidation). ApoB was quantified by laser immunonephelometry (Behring system).

Cell-mediated LDL oxidation was performed by incubating LDL with human ECs under the following conditions. ECs were seeded at a concentration of 4x10⁵ cells/mL in six-multiwell plates (Nunc). When the cells reached subconfluence, they were washed twice and then grown in RPMI-1640 containing native LDL (200 µg apoB per milliliter) at 37°C for various periods of time (or incubation times indicated in the text). At the end of incubation, LDL-containing medium was removed and immediately used for determining the oxidation level (TBARS and REM). The viability of cells was determined by the CFDA test, as indicated below. Copper-mediated LDL oxidation was performed by incubating 2 mg apoB per milliliter LDL (dialyzed overnight against 150 mmol/L NaCl) with 5 µmol/L CuSO₄ for 2 hours at 37°C. LDL oxidation levels were evaluated by monitoring TBARS formation according to the method of Yagi³⁶ and the REM on Hydragel (Sebia).

Cytotoxicity Measurements
The viability of cells was determined to either control the (lack of) toxic effects of cell treatments (ie, photosensitization or treatment with respiratory-chain inhibitors) or evaluate the cytotoxic effects of cell-oxidized LDL on cultured fibroblasts (used as a test system because human fibroblasts do not induce any appreciable LDL oxidation under the conditions employed.)³⁴ Cytotoxicity was evaluated by simultaneously using two tests: trypan blue dye exclusion^{22 37} and CFDA hydrolysis as described by McGinnes et al³⁸ under the conditions previously used.³⁹

Mitochondrial Photosensitization
ECs were grown in RPMI-1640 medium containing 10% FCS (in 150-cm² falcons, Nunc). At subconfluence, the cells were labeled with DASMPI (10 µmol/L in RPMI-1640 for 1 hour), a cell-permeant styrylpyridinium fluorescent indicator that selectively associates with mitochondria.⁴⁰ After being washed twice in RPMI-1640, ECs were irradiated under white light (the falcon was suspended horizontally 15 cm under a 150-W Claudfar lamp) for various times indicated in the text. The medium was removed and replaced with fresh RPMI-1640, and ECs were used for either oxidation experiments, evaluation of the viability of mitochondria, or testing mitochondrial function (evaluation of membrane potential by monitoring rhodamine 123 uptake).^{41 42}

Fluorescence Microscopy of Cells Stained With Mitochondrion-Selective Fluorescent Probes
ECs were incubated with the mitochondrion-selective fluorescent dyes DASMPI (10 µmol/L in phenol red–free RPMI-1640 for 20 minutes) or rhodamine 123 (1 µmol/L in phenol red–free RPMI-1640), washed twice in phenol red–free RPMI-1640, and observed under a microscope equipped with epifluorescence illumination (Leica model Fluovert-FU).

Determination of O₂^- and H₂O₂ Production
Generation of O₂^- by cells was estimated by SOD-inhibitable reduction of ferricytochrome c.⁴³ Cells were grown in six-multiwell culture plates (Nunc). After preconfluent cells were washed with PBS, 1 mL of phenol red–free RPMI-1640 containing 20 µmol/L ferricytochrome c was added and the time course of cytochrome c reduction monitored by reading the absorbance at 550 nm in the presence or absence of SOD (50 µg containing 210 U) at 37°C. O₂^- production was calculated as the difference in absorbance readings between paired dishes incubated with or without SOD; a molar extinction coefficient of 21 000 was used for reduced cytochrome c.⁴³

H₂O₂ production was evaluated by determining the oxidation of scopoletin in the presence of horseradish peroxidase according to Szatrowski and Nathan.⁴⁴ Preconfluent cells grown in 25-cm² culture flasks were carefully washed three times with PBS at 37°C and incubated in 2 mL phenol red–free RPMI-1640 containing 35 µmol/L scopoletin. The reaction was initiated by adding horseradish peroxidase (1 purpurogallin unit per milliliter). Under these conditions, H₂O₂ oxidizes scopoletin to a nonfluorescent compound. Fluorescence quenching was monitored fluorometrically on a Jobin-Yvon spectrofluorometer (model JY3C), and H₂O₂ production was expressed as nanomoles of H₂O₂ produced per hour per milligram of cell protein.

Determination of SOD Activity
SOD activity was measured by using the xanthine–xanthine oxidase/cytochrome c system described by McCord and Fridovitch.⁴³ In brief, the reaction mixture contained 20 µmol/L cytochrome c, 100 µmol/L hypoxanthine, and cell protein (50 µg homogenate prepared in water by disrupting the cells by sonication; three pulses of 10 seconds each in a Soniprep sonicator). The reaction was started by adding xanthine oxidase (a sufficient amount to cause an increase in absorbance at 553 nm of 25 optical density milliunits per minute in the absence of homogenate), and absorbance was continuously recorded for 2 minutes (Kontron Uvicon-930 spectrophotometer). The cyanide-insensitive MnSOD was measured in the presence of 1 mmol/L KCN, Cu-ZnSOD, or cyanide-sensitive SOD and was estimated as the difference between total SOD and MnSOD values.⁴³

Protein concentrations were determined by the procedure of Lowry et al.⁴⁵

	Results

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As expected, the human EC line used was able to oxidize LDL, in agreement with data previously reported in other EC lines except those of bovine origin.^{8 9 10} Under the conditions used, the EC-mediated LDL oxidation was linear up to a 24-hour pulse. The LDL oxidation mediated by human ECs and the subsequent toxicity to human cultured fibroblasts were inhibited by SOD and desferrioxamine but not significantly inhibited by mannitol, L-NAME, or L-NMMA (Table 1). It is noteworthy that the results obtained with various preparations of catalase were rather puzzling. In agreement with the conclusions of Steinbrecher,¹⁹ our data suggest that some commercial preparations of catalase (batch 1) contained heat-stable antioxidants because LDL oxidation was inhibited by batch 1, even after heat treatment at 100°C for 15 minutes, whereas more purified catalase preparations (batches 2 and 3 and extensively purified catalase) did not inhibit LDL oxidation despite their higher enzymatic activity. To investigate the potential role of mitochondrial function in cell-mediated LDL oxidation, we compared the rate of LDL oxidation promoted by normal ECs with that promoted by mitochondria deenergized by either photosensitization or respiratory-chain inhibitors.

View this table: [in this window] [in a new window]   Table 1. Effects of SOD, Catalase, Desferrioxamine, Mannitol, and NO Synthase Inhibitors on EC-Mediated LDL Oxidation

Photosensitization of Mitochondria
Selective photosensitization of mitochondria was performed by using the mitochondrion-selective fluorescent dye DASMPI, which is taken up by and accumulates selectively in the mitochondria by a mechanism that is dependent on the mitochondrial membrane potential.⁴² When cells labeled with DASMPI were irradiated with white light for 3 minutes or longer, we observed dramatic morphological changes in the intracellular distribution of the fluorescent dye (Fig 1). In nonirradiated cells, the fluorescence of mitochondria appeared as a fine, granular network that converged in the perinuclear area, with the nucleus itself, however, being nonfluorescent (Fig 1A through 1C). After photosensitization, the DASMPI-fluorescent cytoplasmic granules (mitochondria) disappeared, whereas the nuclear area became intensely fluorescent and the cytoplasm slightly and diffusely fluorescent (Fig 1D and 1E).

View larger version (63K): [in this window] [in a new window]   Figure 1. Effect of increasing photosensitization times (of irradiation for 0, 1, 2, 3, and 5 minutes, respectively, in panels A through E under the conditions indicated in "Methods") on the intracellular localization of the mitochondria-specific probe DASMPI fluorescence in human ECs. ECs were preincubated with DASMPI (10 µmol/L for 1 hour), then washed twice in RPMI-1640, and photosensitized under white light for 3 minutes under the conditions described in "Methods."

In photosensitized cells, transmission electron microscopy showed structural alterations of the mitochondria, such as swelling, vacuolization, and ballooning and rupture of the mitochondrial membrane (Fig 2). The viability of cells (evaluated by trypan blue dye uptake) irradiated for 3 minutes was only slightly altered, whereas more cells were killed by longer periods of irradiation (Table 2). The data reported in Fig 1 and Table 2 led us to choose a standard irradiation time of 3 minutes, which permitted the mitochondria to deenergize without causing prohibitive levels of immediate cell death. Twenty-four hours after photosensitization, most ECs were still alive (85%) but had not recovered their mitochondrial function, since the distribution of rhodamine 123 was still abnormal (Fig 3).

View larger version (222K): [in this window] [in a new window]   Figure 2. Transmission electron photomicrographs of mitochondria from human ECs. A, Control cells (no photosensitization); B, cells preincubated with DAPSMI and photosensitized for 3 minutes under the conditions referred to in the legend to Fig 1. Bars represent 1 µm.

View this table: [in this window] [in a new window]   Table 2. Cytotoxic Effect of Increasing Time of Photosensitization on ECs Incubated With or Without DASMPI (10 µmol/L)

View larger version (116K): [in this window] [in a new window]   Figure 3. Persistence of mitochondrial dysfunction in photosensitized ECs, as shown by the uptake and intracellular distribution of rhodamine 123. A, Control cells (no photosensitization); B, cells preincubated with DAPSMI and photosensitized for 3 minutes under the conditions referred to in the legend to Fig 1. Then the cells were grown in standard culture medium for 24 hours and incubated with rhodamine 123 as indicated in "Methods."

Mitochondrial Photosensitization of ECs Reduces Their O₂^- Production and LDL Oxidation
The O₂^- production by photosensitized ECs was reduced by 50% in comparison with controls, ie, normal (unlabeled and nonirradiated) ECs or cells labeled with DASMPI but not irradiated (Fig 4). Consistent with the reduced O₂^- production, the level of TBARS in LDL oxidized by photosensitized ECs was significantly reduced when compared with cell-induced LDL oxidation by nonphotosensitized controls (Fig 5A). The REM as determined by electrophoresis on Hydragel was increased, as were the lipid peroxidation indices: 1.8 and 1.2 for LDL oxidized by nonphotosensitized and photosensitized cells, respectively. The biological effect of cell-oxidized LDL was tested by evaluating its toxicity to cultured human fibroblasts. As shown in Fig 5B, the cytotoxicity of LDL oxidized by photosensitized ECs was reduced in the same proportion as were the TBARS levels.

View larger version (22K): [in this window] [in a new window]   Figure 4. O2- production in DASMPI-photosensitized (filled symbols) and control (empty symbols) ECs. Control 1, no DASMPI, no irradiation (empty circles); control 2, no DASMPI, irradiation (empty triangles); and control 3, DASMPI, no irradiation (empty squares). In the assays (filled circles), ECs were preincubated with DASMPI (10 µmol/L for 1 hour) and after being washed were photosensitized for 3 minutes under the conditions referred to in the legend to Fig 1. Then the cells were washed and grown in fresh culture medium. O2- production was determined 12 hours later as indicated in "Methods." Values are mean±SEM of four separate experiments.

View larger version (23K): [in this window] [in a new window]   Figure 5. LDL oxidation (A) by DASMPI-photosensitized and control ECs and its subsequent toxicity (B) to cultured fibroblasts. ECs (photosensitized or not) were incubated for 12 hours in the presence of native LDL (200 µg/mL culture medium). At the end of this 12-hour period, oxidation levels were evaluated immediately by monitoring TBARS formation (expressed as nmol TBARS/mg apoB; A). The subsequent toxicity was evaluated by transferring the culture medium to cultured fibroblasts and estimating their viability 48 hours later by using the CFDA test, expressed as a percent (fluorescence arbitrary units) of the untreated control (B). Values are mean±SEM of three separate experiments.

Effect of Respiratory-Chain Inhibitors and CCCP on O₂^- Production and LDL Oxidation by ECs
To confirm the involvement of mitochondrial function in O₂^- production by ECs, we tested the comparative effects of two inhibitors of the mitochondrial respiratory chain, antimycin A and rotenone (which act at the level of complex I/III and IV, respectively) and the uncoupler CCCP. The inhibitor concentrations were chosen to induce mitochondrial deenergization, as shown by the alteration in rhodamine 123 uptake (Fig 6), without any major alteration of the viability of ECs incubated for 24 hours in the presence of the inhibitors (Table 3). The concentrations of antimycin A, rotenone, and CCCP significantly reduced cellular production of O₂^- (Fig 7), cell-mediated oxidation of LDL (Fig 8A), and its subsequent toxicity to fibroblasts (Fig 8B). It is noteworthy that when antimycin A and CCCP were added simultaneously, the production of O₂^- "recovered" to nearly its initial level (Fig 7), as did the level of LDL oxidation (Fig 8). ECs also produced H₂O₂ (data not shown), the level of which was influenced similar to that of O₂^- by mitochondrial inhibitors. However, as reported above, (due to the lack of effect of purified catalase and mannitol), H₂O₂ and OH do not seem to be involved in LDL oxidation in this experimental model system.

View larger version (89K): [in this window] [in a new window]   Figure 6. Uptake and intracellular distribution of rhodamine 123 in human ECs preincubated for 24 hours with mitochondria inhibitors. First panel, Control cells (no inhibitor); second panel, 10 µmol/L antimycin A; third panel, 10 µmol/L rotenone; and fourth panel, 50 µmol/L CCCP. After this 24-hour preincubation, cells were incubated with rhodamine 123 (as indicated above) and immediately examined by fluorescence microscopy.

View this table: [in this window] [in a new window]   Table 3. Effect of Mitochondrial Respiratory-Chain Inhibitors and the CCCP Uncoupler on Viability of Human ECs

View larger version (24K): [in this window] [in a new window]   Figure 7. O2- production by human ECs preincubated for 24 hours with mitochondria inhibitors under the conditions referred to in the legend to Fig 6. 0, Control cells (no inhibitor); A, 10 µmol/L antimycin A; R, 10 µmol/L rotenone; C, 50 µmol/L CCCP; and A+C, 10 µmol/L antimycin A and 50 µmol/L CCCP. At the end of this 24-hour preincubation, O2- production was determined as indicated in "Methods." Values are mean±SEM of four separate experiments.

View larger version (22K): [in this window] [in a new window]   Figure 8. LDL oxidation by human ECs treated by mitochondria inhibitors (under the conditions referred to in the legends to Figs 6 and 7). A, 10 µmol/L antimycin A; R, 10 µmol/L rotenone; C, 50 µmol/L CCCP; A+C, 10 µmol/L antimycin A and 50 µmol/L CCCP; and subsequent toxicity to cultured fibroblasts. ECs (preincubated) were incubated for 12 hours in the presence of mitochondria inhibitors and native LDL (200 µg/mL culture medium). At the end of this 12-hour period, (A) LDL oxidation was evaluated immediately by determining TBARS levels (expressed as nmol/mL culture medium of 106 cells) and (B) the subsequent toxicity was evaluated by transferring the culture medium to cultured fibroblasts and determining the CFDA index 48 hours later (results are expressed as percent of control). Values are mean±SEM of three separate experiments.

MnSOD Activity in Mitochondria-Deenergized Cells
Because O₂^- production by ECs seems to be related to mitochondrial function, we examined the activity of SOD, which is known to be inducible⁴⁶ by oxidizing agents.³² As reported in Table 4, MnSOD represented 15% to 20% of the total cellular SOD in resting cells. This level of activity was significantly increased in photosensitized cells but was in the normal range for cells incubated for 12 hours with respiratory-chain inhibitors or CCCP. These latter results suggest that the decreased O₂^- production cannot be attributed in all cases to the induction of MnSOD.

View this table: [in this window] [in a new window]   Table 4. Effects of Mitochondrial Respiratory-Chain Inhibitors, the CCCP Uncoupler, and Photosensitization on Cellular SOD Activity

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Cultured ECs are known to be able to oxidize LDL through free radical–dependent mechanisms,^{8 9 10} but the source and precise mechanism of production of these free radicals is poorly understood.^{14 15 16 17 18 19 20 21 22 23 24 25 28 29} To our knowledge, the potential role of mitochondrial function in the production of O₂^- and the subsequent LDL oxidation by ECs is not known. The data reported herein allow us to establish a clear correlation between mitochondrial function (high membrane potential) and the cellular output of reactive oxygen species and subsequent LDL oxidation by human cultured ECs. This conclusion is supported by the concomitant decrease in O₂^- production and LDL oxidation by cells in which mitochondrial function has been altered by selective photosensitization or mitochondrial inhibitors.

Human CRL-1998 ECs grown in RPMI induced a significant level of LDL oxidation, whereas under the same conditions, the bovine EC line GM-7372A did not induce LDL oxidation (data not shown), in agreement with the data of Steinbrecher.¹⁹ Despite the fact that RPMI contains no detectable levels of iron or copper ion (according to the manufacturer and in agreement with Reference 21²¹ ), desferrioxamine inhibited EC-mediated LDL oxidation. This result suggests that trace amounts of transition metal ions were either probably present in the RPMI or LDL solution or released from the cells. This latter mechanism, ie, enhancement of LDL oxidation by iron released from cells (enriched with iron through erythrophagocytosis), has been recently reported in macrophages.⁴⁷ The inhibitory effect of exogenous SOD strongly suggests that LDL oxidation promoted by human ECs is mediated at least in part by O₂^-, in agreement with previous reports on rabbit endothelial¹⁹ or other vascular¹⁸ cells but in contrast with the findings reported in Reference 48⁴⁸ . Our data are also consistent with those of Bedwell et al,⁴⁹ who reported that O₂^- is able to oxidize LDL only in the presence of a transition metal.

Because the EC line used generated a significant level of H₂O₂ and transition metal ions seem to have been present (as discussed above), it is possible that the Fenton reaction may have occurred, leading to the formation of OH^- radicals, which are known to be able to oxidize LDL.⁴⁹ However, under the experimental conditions used, this hypothetical mechanism of LDL oxidation is not supported by the experiments with catalase and mannitol. Mannitol (an OH^- scavenger) did not significantly decrease the oxidation rate, in agreement with previously reported data.^{19 48 50} Similarly, purified catalase did not inhibit LDL oxidation (in contrast to more crude preparations, which contained a heat-stable antioxidant compound), consistent with the findings in Reference 19¹⁹ . Therefore, we suggest that under the experimental conditions used, H₂O₂ and OH^- do not seem to play a major role in EC-mediated LDL oxidation. Similarly, NO and peroxynitrite do not seem to be involved in cell-mediated LDL oxidation, as shown by the lack of inhibition of LDL oxidation by L-NAME and L-NMMA, which were used at effective concentrations that effectively inhibit NO synthase.

The assumption that mitochondrial function plays a role in EC-mediated LDL oxidation is supported by the observation that O₂^- production and subsequent LDL oxidation by intact ECs were considerably decreased under conditions that altered mitochondrial function (photosensitization and the use of uncoupler or mitochondrial respiratory-chain inhibitors). It is noteworthy that despite mitochondrial deenergization, the ECs did not die immediately, possibly because they are able to survive short periods of anaerobic conditions (eg, they can survive for several days in the presence of respiratory-chain inhibitors; data not shown).

Under the mild conditions used here, DASMPI-mediated photosensitization of the mitochondria induced a disturbance in mitochondrial function, leading to the loss of mitochondrial membrane potential as assessed by changes in the uptake and intracellular distribution of rhodamine 123.⁴¹ During the first 24 hours after mitochondrial photosensitization, most of the cells were still alive but exhibited a dramatic decrease in O₂^- production, LDL oxidation, and subsequent cytotoxic effects. Similarly, O₂^- production as well as LDL oxidation by intact ECs were inhibited by the respiratory-chain inhibitors rotenone and antimycin and the uncoupler CCCP. It is interesting that when antimycin and CCCP were added simultaneously, O₂^- and H₂O₂ production were maintained at near-normal levels (Figs 7 and 8), despite mitochondrial deenergization. This latter result demonstrates that under these conditions, the inhibition of O₂^- production and LDL oxidation in cells treated with mitochondrial inhibitors does not result from a nonspecific toxic effect. Moreover, it is also interesting that inhibition of LDL oxidation by mitochondrial inhibitors was not due to a nonspecific antioxidant effect of these inhibitors, because at the concentrations used, they did not inhibit copper-induced LDL oxidation (Table 5).

View this table: [in this window] [in a new window]   Table 5. Effect of Mitochondrial Respiratory-Chain Inhibitors and the CCCP Uncoupler on Copper-Mediated LDL Oxidation

All the data reported and discussed above strongly suggest that mitochondrial function is involved in O₂^-/H₂O₂ production and LDL oxidation by intact ECs. However, the link between mitochondrial function and output of O₂^-/H₂O₂ remains largely hypothetical. In fact, two hypotheses may be proposed: Mitochondrial O₂^-/H₂O₂ production is involved in the output of O₂^-/H₂O₂ either (1) directly: or (2) indirectly, through more complex and subtle mechanisms regulating other (extramitochondrial) cellular systems that also produce O₂^-/H₂O₂.

In the first hypothesis (mitochondrial O₂^-/H₂O₂ production is directly involved in the output of O₂^-/H₂O₂), several points remain unexplained. Rotenone inhibits complex I (NADH dehydrogenase) and at the same time inhibits mitochondrial oxygen consumption and O₂^- and H₂O₂ production.^{51 52} Antimycin exerts its inhibitory effect on complex III by inhibiting electron flow at the site of electron leakage; thus, mitochondrial oxygen consumption is inhibited but O₂^- and H₂O₂ production is increased in isolated mitochondria.⁵² By contrast, in intact living cells, antimycin at relatively high concentrations has been shown to inhibit O₂^- production,^{53 54} in agreement with the data reported in this article. The reason for this discrepancy (between antimycin's effects on isolated mitochondria and those in intact cells) is still unknown, but it does not seem to result from induction of MnSOD (Table 4). The uncoupler CCCP induces the loss of mitochondrial membrane potential and markedly inhibits O₂^- production, in agreement with the inhibitory effect of the FCCP uncoupler on H₂O₂ production in liver slices.⁵⁵

In the second hypothesis (mitochondrial function or O₂^-/H₂O₂ production is indirectly involved in the output of O₂^-/H₂O₂), mitochondrial depolarization in principle can induce ATP depletion, alter redox equilibria and calcium homeostasis,⁵⁶ and subsequently cause a variety of effects on cell metabolism. In turn, these changes in intracellular homeostasis may affect the production of radical species somewhere else in the cell and finally alter O₂^-/H₂O₂ output. However, it should be noted that O₂^-/H₂O₂ output was not directly correlated with mitochondrial deenergization, as shown by the different effect of mitochondrial inhibitors used alone or in combination with CCCP. An alternative hypothesis postulates that O₂^- may act as a second messenger.^{57 58} Therefore, mitochondrial O₂^- may participate in regulating other enzyme systems involved in O₂^- production, which are located elsewhere in the cell.

In conclusion, the data reported here strongly support the hypothesis that in resting ECs, mitochondrial respiration plays a role in the generation of O₂^-, which is involved directly or indirectly in LDL oxidation and atherogenesis. This source of O₂^- may take part in the oxidative stress and inflammatory response involved in atherogenesis.⁵⁹ Because atherosclerosis clearly progresses with age,⁶⁰ the reported data agree with the assertion that the mitochondria appear to be a source of oxidative lesions that accumulate with age.⁶¹

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein CCCP = carbonylcyanide-m-chlorophenylhydrazone CFDA = carboxyfluorescein diacetate acetoxymethylester DASMPI = 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide EC = endothelial cell FCS = fetal calf serum L-NAME = L-nitroarginine methylester L-NMMA = N-[methylamidino]-L-ornithine OxLDL = oxidized LDL REM = relative electrophoretic mobility TBA(RS) = thiobarbituric acid (–reactive substances)

	Acknowledgments

 
The financial support of INSERM (CJF 9206), Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, University Paul Sabatier Toulouse-3, and of the European Communities (PL 931790) is gratefully acknowledged. The authors wish to thank C. Mora, J. Dumoulin, and J.C. Thiers for the excellent technical assistance.

Received March 6, 1996; accepted October 30, 1996.

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