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Am J Physiol Heart Circ Physiol 285: H2298-H2308, 2003. First published July 24, 2003; doi:10.1152/ajpheart.00508.2003
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Oxidized low-density lipoprotein and 15-deoxy-{Delta}12,14-PGJ2 increase mitochondrial complex I activity in endothelial cells

Erin K. Ceaser,1 Anup Ramachandran,1 Anna-Liisa Levonen,1 and Victor M. Darley-Usmar1,2

1Department of Pathology and 2Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Submitted 2 June 2003 ; accepted in final form 21 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Oxidized lipids are capable of initiating diverse cellular responses through both receptor-mediated mechanisms and direct posttranslational modification of proteins. Typically, exposure of cells to low concentrations of oxidized lipids induces cytoprotective pathways, whereas high concentrations result in apoptosis. Interestingly, mitochondria can contribute to processes that result in either cytoprotection or cell death. The role of antioxidant defenses such as glutathione in adaptation to stress has been established, but the potential interaction with mitochondrial function is unknown and is examined in this article. Human umbilical vein endothelial cells (HUVEC) were exposed to oxidized LDL (oxLDL) or the electrophilic cyclopentenone 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2). We demonstrate that complex I activity, but not citrate synthase or cytochrome-c oxidase, is significantly induced by oxLDL and 15d-PGJ2. The mechanism is not clear at present but is independent of the induction of GSH, peroxisome proliferator-activated receptor (PPAR)-{gamma}, and PPAR-{alpha}. This response is dependent on the induction of oxidative stress in the cells because it can be prevented by nitric oxide, probucol, and the SOD mimetic manganese(III) tetrakis(4-benzoic acid) porphyrin chloride. This increased complex I activity appears to contribute to protection against apoptosis induced by 4-hydroxynonenal.

oxidized lipid; reactive oxygen and nitrogen species; peroxisome proliferator-activated receptors; 4-hydroxynonenal

MITOCHONDRIAL DYSFUNCTION is known to underlie a number of pathogenic processes, including cardiac ischemiareperfusion and neurodegenerative diseases (7, 33). Interestingly, little is known of the changes in mitochondrial function that occur during the pathogenesis of atherosclerosis, although some early studies suggested that a progressive mitochondrial dysfunction occurs during the development of the disease (16, 19, 44, 45). Mitochondrial function is now known to play a role in apoptosis and other aspects of signal transduction, and this has renewed interest in its potential role in the response of the endothelium to the oxidative stress associated with the development of atherosclerosis (2, 3, 33, 34). Indeed, it has been demonstrated that mitochondrial DNA (mtDNA) is particularly susceptible to damage mediated by reactive oxygen (ROS) and nitrogen species (RNS) in both endothelial and vascular smooth muscle cells (2, 3). Analysis of the functional changes that occur during the mitochondrial dysfunction associated with neurodegenerative syndromes or ischemia-reperfusion have often indicated the particular susceptibility of complex I in the electron transport chain to oxidative modification (7, 20, 33).

Very little is known about the role of mitochondrial proteins in the adaptive responses of the cell to nontoxic concentrations of ROS or RNS. This is particularly important in chronic diseases such as atherosclerosis because we (18, 26, 28, 34) and others (12, 29) have demonstrated that impairment of mitochondrial function increases the susceptibility of endothelial cells to apoptosis. Interestingly, although the etiology of atherosclerosis is characterized by an increase in ROS, RNS, and lipid oxidation products, their impact on mitochondrial function has not been addressed (5, 14).

The response of vascular cells to lipid oxidation products appears to have two components. The first of these is initiated by exposing cells to low concentrations of oxidized lipids and involves the activation of intracellular cytoprotective pathways. This enhances cytoprotection against a secondary oxidative challenge. For example, our group (26) has shown that oxidized LDL (oxLDL) or 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) can protect against apoptosis induced by redox cycling xenobiotics or the toxic aldehyde 4-hydroxynonenal (4-HNE). The mechanisms involved are only now emerging and include the activation of several signal transduction pathways, including the MAP kinases (17). An interesting example is provided by 15d-PGJ2, which is a potent peroxisome proliferator-activated receptor (PPAR)-{gamma} agonist but also acts independently of this receptor, to induce pathways that at low concentrations are protective and at high concentrations induce apoptosis (26). It has been shown that other PPAR-{gamma} agonists, such as the thiazolidinediones, can modulate endothelial cell growth and inhibit atherosclerotic lesion formation in apolipoprotein E –/– mice and that PPAR-{alpha} receptors play a central role in the {beta}-oxidation of fatty acids (4, 9, 15).

Interestingly, in dopaminergic neurons it has been shown that downregulation of the expression of glutamate-cysteine ligase, the rate-limiting enzyme for GSH synthesis, leads to a selective decrease in complex I activity (23). It is generally thought that the loss of complex I activity associated with GSH depletion results from the oxidation of protein thiols, because it can be reversed by thiol reductants (1). Recent studies support this concept, demonstrating that S-glutathionylation of complex I decreases its activity and increases ROS formation (41). Maintenance of mitochondrial GSH levels is linked to mitochondrial respiration through the actions of a glutathione reductase, which obtains NADPH from a transhydrogenase driven by the mitochondrial membrane potential or the mitochondrial isocitrate dehydrogenase. This indicates a direct requirement for functional complex I activity for the organelle to respond to oxidative stress. We hypothesized that noncytotoxic concentrations of oxidized lipids modulate complex I activity in an ROS-dependent mechanism.

In the present study using endothelial cells we determined the effect of oxLDL and pure preparations of single lipid components on the induction of GSH and complex I. In this report we show that the mechanism of induction of complex I by oxidized lipids is not due to an increase in intracellular GSH or activation of the PPAR-{alpha} or PPAR-{gamma} receptors. The mechanism does require cytoplasmic protein synthesis and involves the formation of ROS or RNS as mediators. The implications of these findings for the response of the endothelium to oxidative stress are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Coenzyme Q1, cytochrome c, NADH, acetyl-CoA, fat-free BSA, rotenone, oxaloacetate, MCDB 131 basal media, and DTNB were purchased from Sigma. Prostaglandins and arachidonic acid were purchased from Cayman Chemicals and PPAR agonists from BioMol. All other reagents used were of analytical grade.

Cell preparation and culture. Human umbilical vein endothelial cells (HUVEC) were purchased at passage 1 from the Endothelial Core Center (Dr. Francois Booyse), University of Alabama at Birmingham. Cells were maintained (37°C, 5% CO2) in MCDB 131 containing 2% FBS, 10 ng/ml human (h)EGF, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and 12 µg/ml bovine brain extract. Cells were used between passages 4 and 8 as confluent monolayers.

Isolation of lipoproteins and oxidation. Differential centrifugation was used to isolate LDL from plasma as previously described (10). The LDL was oxidized by adding 20–100 µM CuSO4 per 2 mg/ml LDL, followed by incubation at 37°C. The extent of oxidation was characterized by measurement of relative electrophoretic mobility (REM) and lipid peroxides.

Assay of respiratory complex activities and citrate synthase. Complex I activity was determined with ~50–100 µg of lysate by following the rotenone-sensitive oxidation of NADH (100 µM) initiated by coenzyme Q1 (50 µM). Basal complex I activity varied from ~30 to 90 nmol · min–1 · mg–1 but was consistent within each experimental group to within 10–15% error. Cytochrome-c oxidase was measured as the oxidation of ferrocytochrome c. Citrate synthase activity was determined as previously described (31). Basal citrate synthase values varied from 140 to 175 nmol · min–1 · mg–1 but remained constant within a 5–10% variation during a single experiment.

Total glutathione measurements. Total glutathione (GSH + GSSG) was determined with a spectrophotometric assay as previously characterized (43). Cells were lysed in PBS buffer containing 0.1% Triton and added to a solution containing PBS, Triton buffer, 600 µM DTNB, 200 µM NADPH, and 10 µg of glutathione reductase. The reaction was monitored for the rate of formation of thionitrobenzoate at 412 nm. A standard curve generated with authentic GSH was used to convert the rates to total GSH concentration.

Blue native electrophoresis, Western blotting, and second-dimension SDS-PAGE. For visualization of respiratory complexes, mitochondria were isolated by differential centrifugation from HUVEC after treatment with 15d-PGJ2 (5 µM) for 16 h. The samples were then subjected to blue native gel electrophoresis as described previously (6) and stained with Coomassie blue. To confirm the identity of complex I, parallel experiments were conducted and gels were transferred onto nitrocellulose after a 20-min equilibration in transfer buffer (containing 20% methanol, 0.05% SDS). The membrane was then washed in transfer buffer containing SDS (1%) and {beta}-mercaptoethanol (10 mM) for 20 min to completely denature proteins. This was followed by blocking in milk powder and probing with an antibody to complex I (30-kDa subunit). For the second-dimension SDS-PAGE, the band corresponding to complex I was excised from the blue native gel and subjected to a 10–18% gradient SDS-PAGE as described previously (6). The gel was then stained with Coomassie blue to visualize the individual subunits, followed by densitometry to determine relative band intensities.

Annexin V binding assays. Apoptosis was measured by labeling the cells with annexin V conjugated to FITC and propidium iodide followed by fluorescence-activated cell sorting (FACS) analysis. HUVEC were grown to confluence in six-well plates and then treated. Cells were then detached with trypsin-EDTA, and after being washed with PBS they were resuspended in annexin V binding buffer containing 0.5 ng of annexin V-FITC and 2.5 ng of propidium iodide. Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA) with WINMDI 2.8 software (Scripps Research Institute Cytometry Software Page; Ref. 37).

Protein assay. The concentration of protein for all samples was determined by the Bradford protein assay with reagents purchased from Bio-Rad and used to normalize all enzyme activities.

Statistical analysis of data. Biochemical experiments were performed in triplicate and repeated two or three times. For all experiments, either a Student's t-test or an ANOVA was used to determine significance (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of oxLDL on endothelial cell mitochondria and GSH. In the first series of experiments the effect of oxLDL on complex I activity and GSH in endothelial cells was determined. Confluent endothelial cells were exposed to oxLDL (0–200 µg/ml) with a REM of 1.8–2.2 for a period of 0–24 h. After treatment, a cell lysate was prepared and the enzymatic activities of complexes I and IV and citrate synthase were measured spectrophotometrically. It was found that complex I activity increased by ~80% after exposure to oxLDL (75 µg/ml) over a period of 16 h (Fig. 1A) and was essentially maximal at concentrations of oxLDL >75 µg/ml (Fig. 1B). The total GSH content of the endothelial cells was determined under these same conditions. As described previously, oxLDL induces GSH synthesis in endothelial cells (26, 28) (Fig. 1C). However, it is interesting to note that the concentration dependence for induction of complex I activity and GSH are different. At 150 µg/ml, oxLDL induction of complex I activity is unchanged, whereas total GSH is returned to control levels (Fig. 1C).



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  		Fig. 1. Effects of oxidized lipids on complex I activity and GSH. Human umbilical vein endothelial cells (HUVEC) were treated with 75 µg/ml of oxidized LDL (oxLDL) for various times and then assayed for complex I activity (A) or treated with varying concentrations of oxLDL for 16 h (B) or increasing concentrations of oxLDL (C) and assayed for GSH. All data are means ± SE; n = 3. *P < 0.05, **P < 0.01 compared with control.

 

In the next series of experiments, the effects of ox-LDL on complex IV and citrate synthase activity were determined. Citrate synthase was selected as a mitochondrial matrix enzyme known to be relatively insensitive to the effects of oxidants and complex IV as an inner mitochondrial membrane protein coded for by both nuclear and mtDNA. In this case the basal level of complex I activity was lower than in the previous experiment and the induction of complex I activity in response to oxLDL was about fourfold greater than that shown in Fig. 1. We have noted that the basal complex I activity and degree of induction in response to oxLDL vary between the individual donors for the HUVEC preparation for reasons that remain unclear. It was seen that cytochrome-c oxidase and citrate synthase activity showed no significant change on exposure to oxLDL (Fig. 2). The addition of neither native LDL nor copper-diethylenetriamine pentaacetic acid at the same concentration as used in the oxLDL preparation had any significant effect on the activity of any of the mitochondrial complexes (Fig. 2).



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  		Fig. 2. Complex I, complex IV, and citrate synthase assays in HUVEC. HUVEC were treated for 24 h with 75 µg/ml native LDL (nLDL) or oxLDL. Cell lysate was then assayed for complex I activity (A), complex IV activity (B), and citrate synthase activity (C). All data are means ± SE; n = 3. *P < 0.05 compared with control. DTPA, diethylenetriamine pentaacetic acid; k, rate constant.

 

Effects of specific oxidized lipids on respiratory chain activity. oxLDL is an extremely complex mixture of lipid and protein adducts. In the next series of experiments selected oxidized lipids, either present in oxLDL or structurally analogous to the electrophilic lipid oxidation products, were tested. It was found that the lipid peroxide derived from linoleic acid, 13(s)-hydroperoxy-(9Z,11E)-octadecadienoic acid (25 µM), or the aldehyde 4-HNE (10 µM) had little or no effect on complex I activity (result not shown). However, the electrophilic cyclopentenone prostaglandin 15d-PGJ2 induced complex I activity approximately twofold after treatment over a period of 5–15 h (Fig. 3A). Again in this series of experiments some variation (1.5- to 2-fold) in the degree of induction of complex I activity occurred in response to exposure to 15d-PGJ2. As shown in Fig. 4, compounds that can be converted to, or contain, the {alpha},{beta}-unsaturated ketone (15d-PGJ2, PGA1, PGD2, PGJ2) significantly increased complex I activity and GSH levels, whereas PGE2, which does not have this functional group and is not converted to an electrophilic lipid, had no significant effect on either complex I activity or GSH. Under identical conditions it has been shown that the ratio of GSH to GSSG on exposure to 15d-PGJ2 increases over a 16-h time course (26).



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  		Fig. 3. Time course and concentration dependence of HUVEC treated with 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) and peroxisome proliferator-activated receptor (PPAR) agonists. HUVEC were treated for the times shown with 2.5 µM 15d-PGJ2 (A) or treated for 24 h with varying concentrations of 15d-PGJ2 (B), the PPAR-{gamma} agonists troglitazone (5 µM) and ciglitazone (5 µM) (C), or the PPAR-{alpha} agonists fenofibrate (25 µM) and WY-14643 (40 µM) (D). All data are means ± SE; n = 3. *P < 0.01 compared with control.

 


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  		Fig. 4. Effect of prostaglandins on complex I activity and GSH. HUVEC were treated for 24 h with 15d-PGJ2, PGA2, PGD2, PGE2, and PGI2. All data are means ± SE; n = 3. *P < 0.005 compared with vehicle-treated control.

 

PPAR-{gamma} does not mediate upregulation of complex I. As mentioned above, it is known that 15d-PGJ2 is a potent PPAR-{gamma} agonist. It was hypothesized that the induction of complex I activity was mediated through this pathway. As shown in Fig. 3C, exposure of endothelial cells to the PPAR-{gamma} agonists ciglitazone and troglitazone resulted in little or no change in complex I activity compared with the increase induced by 15d-PGJ2 (Fig. 3B). PPAR-{alpha} agonists have also been shown to modulate mitochondrial function and protect against atherosclerosis. In the next series of experiments the potential role of PPAR-{alpha} in the induction of complex I activity was examined by exposing the endothelial cells to fenofibrate and WY-14643. As seen in Fig. 3D, neither compound had any significant effect on complex I activity.

Increase in complex I activity induced by 15d-PGJ2 and effects on GSH. It has been proposed that increased GSH could lead to increased complex I activity (1). To test this hypothesis, several approaches were taken to modulate GSH levels over a wide range and then complex I activity was measured as summarized in Table 1. In this series of experiments L-buthionine sulfoxamine (BSO) was used to inhibit the rate-limiting enzyme for GSH synthesis, glutamate-cysteine ligase, or acivicin to inhibit {gamma}-glutamyltranspeptidase, which also attenuates GSH synthesis. HUVEC were pretreated for 30 min with BSO (25 µM) or acivicin (5 µM) followed by an 18-h cotreatment with 15d-PGJ2 (2.5 µM). Under these conditions a concentration of 15d-PGJ2 was selected that was below the threshold cytotoxicity in the presence or absence of BSO. As expected, 15d-PGJ2 increased GSH levels. This was completely inhibited (95%) by BSO but only had a slight, but not significant, effect on complex I induction (Table 1). In the case of acivicin, GSH levels were decreased 32% and the 1.6-fold increase in complex I activity was not affected (Table 1). Together these data indicate that total cellular GSH can vary over a wide range and have relatively little effect on the induction of complex I activity by 15d-PGJ2.


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  		Table 1. Effect of inhibiting GSH synthesis on induction of complex I activity

 

Increase in complex I activity after treatment with oxidized lipids requires nuclear protein synthesis. To determine whether cytoplasmic or mitochondrial protein synthesis is necessary for the increase in complex I activity, HUVEC were treated for 24 h with 15d-PGJ2 (2.5 µM) in combination with cycloheximide, an inhibitor of cytoplasmic protein synthesis, and/or chloramphenicol, an inhibitor of mitochondrial protein synthesis (Fig. 5). Increased complex I activity in response to 15d-PGJ2 was not affected by chloramphenicol but was decreased by cycloheximide. It is important to note that under these conditions complex I activity is only inhibited after more extended exposure to chloramphenicol (result not shown). Together, these data suggest that synthesis of the nuclear encoded proteins is essential for the induction of complex I activity. To determine whether changes in complex I protein levels were associated with the increase in activity induced by oxLDL, Western blots were performed with the antibody to the nuclear encoded 39-kDa subunit of complex I. As a control, subunit 1 of complex IV was also measured. Neither the levels of the 39-kDa subunit of complex I nor subunit I of complex IV changed on exposure to oxLDL (Fig. 5B).



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  		Fig. 5. Induction of complex I activity requires protein synthesis. A: HUVEC were pretreated with the protein synthesis inhibitors chloramphenicol (CAP; mitochondrial synthesis) and cycloheximide (Cyclo; nuclear synthesis) and then cotreated with 15d-PGJ2. Cells were assayed for complex I activity after 24 h. B: HUVEC treated with 75 µg/ml oxLDL were lysed and subjected to Western blot analysis. The blot was probed with an antibody to the 39-kDa subunit of complex I and an antibody to subunit 1 of complex IV. Mito, mitochondria. All data are means ± SE. *P < 0.05 compared with 15d-PGJ2.

 

To investigate whether treatment with 15d-PGJ2 results in alterations at the protein level in complex I, blue native PAGE was used, which allows measurement of the amounts of the respiratory complexes in their native state. In this experiment, cells were exposed to 15d-PGJ2 for 16 h before being washed and a mitochondrially enriched fraction was prepared. These samples were then separated by blue native PAGE followed by Coomassie blue staining to visualize the complexes. As seen in Fig. 6, treatment with 15d-PGJ2 did not result in significant alteration in levels of native complex I. However, this enzyme is a multi-subunit protein complex composed of 45 subunits, and alterations in a few constituent subunits may not be evident when the complex is examined in the native state. Hence, a second-dimension denaturing SDS-PAGE was performed to separate individual subunits. Western blotting of the first dimension with an antibody against the 30-kDa subunit of complex I confirmed the position of the complex on the blue native gel. This band was excised and separated on an SDS-PAGE gradient gel, followed by staining and densitometry. Although it cannot be confirmed by this technique that all the bands seen on the second dimension are complex I subunits, relative band intensity determination did not illustrate any significant changes after treatment with 15d-PGJ2 compared with control, indicating that the increase in complex I activity is independent of protein levels. Together these data suggest that the induction of a protein encoded by nuclear DNA, but not necessarily a complex I subunit, causes the increased activity of complex I on exposure to 15d-PGJ2.



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  		Fig. 6. Characterization of complex I in HUVEC after treatment with 15d-PGJ2. A: mitochondria isolated from HUVEC after treatment with 15d-PGJ2 were separated on blue native PAGE gels, followed by staining with Coomassie blue to visualize the complexes. B: the blue native gels were Western blotted in parallel experiments and probed with an antibody to complex I (30-kDa subunit) to confirm position of the complex I on the first-dimension gel. C: relative band intensity expressed as fold change compared with control for complex I after densitometry. D: the band corresponding to complex I was excised from the blue native gel before staining and then separated on a denaturing SDS-PAGE 10–18% gradient gel to elucidate the individual subunits, followed by Coomassie blue and densitometry to determine relative band intensities (E).

 

Antioxidants inhibit induction of complex I activity. The exposure of 15d-PGJ2 is thought to result in transient oxidative stress at the level of the mitochondria, leading to further lipid peroxidation in cells. (25) In turn, we hypothesized that the balance between NO and lipid-derived oxidants or superoxide is critical in controlling the pathways that lead to the induction of complex I activity. As such, it would be anticipated that peroxyl radical scavengers would prevent this response. To test this hypothesis, HUVEC were cotreated with oxLDL (75 µg/ml) and either probucol (10 µM) or the NO donor diethylenetriamine NONOate (250 µM) (Fig. 7A). Cotreatment with either probucol or NO inhibited the induction of complex I activity, implicating a role for ROS in this process. The cotreatments with these antioxidants attenuated the induction of GSH levels by ~50% (Fig. 7B). As a further test of the role of ROS in inducing complex I activity, HUVEC were treated with 15d-PGJ2 to induce complex I activity and cotreated with manganese(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). Under these conditions MnTBAP resulted in an attenuation of the increased complex I activity after 15d-PGJ2 exposure, independent of changes in the induction of GSH synthesis (Fig. 7, C and D).



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  		Fig. 7. Antioxidants inhibit the induction of complex I activity. A and B: HUVEC were treated for 24 h with 75 µg/ml oxLDL in conjunction with either probucol (10 µM) or the NO donor diethylenetriamine (DETA) NONOate (250 µM). C and D: HUVEC were treated with 5 µM 15d-PGJ2 and 15 µM manganese(III) tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). Cells were then assayed for complex I activity and GSH levels. All data are means ± SE; n = 3. *P < 0.05 compared with control. **P < 0.05 compared with OXLDL. §P < 0.05 compared with MnTBAP.

 

MnTBAP has the capacity to scavenge H2O2, peroxynitrite, or superoxide. These data could be consistent with a role of these oxidants in inducing complex I activity. To determine the role of RNS in the induction of complex I activity, HUVEC were treated for 24 h with the peroxynitrite donor 3-morpholinosydnonimine (SIN-1, 250 µM–1 mM) (Fig. 8). Complex I activity was increased almost twofold with all concentrations, suggesting that a reactive nitrogen species, peroxynitrite, capable of initiating lipid peroxidation could play a role in the induction of complex I activity.



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  		Fig. 8. 3-Morpholinosydnonimine (SIN-1) causes an induction of complex I activity. HUVEC were treated for 24 h with increasing concentrations of the peroxynitrite donor SIN-1. Cells were then assayed for complex I activity. All data are means ± SE; n = 3. *P < 0.05 compared with control.

 

Induction of complex I activity is cytoprotective. To determine whether the observed induction of complex I activity could contribute to protection of the cells against oxidative stress HUVEC were pretreated for 24 h with 2.5 µM 15d-PGJ2 and then treated for 16 h with a complex I inhibitor. In the initial experiments rotenone was used but was found to give inconsistent inhibition of complex I because of interference with components in the cell medium. As an alternative the irreversible flavoprotein inhibitor diphenyleneiodonium (DPI) was titrated into the cells and the inhibition of complex I was determined (Fig. 9A). It was found that 10 µM DPI inhibited complex I activity by ~50%. In the second part of the experiment, cells were exposed to a cytotoxic concentration of 4-HNE in the absence or presence of DPI and then subjected to FACS analysis to determine cell death (Fig. 9, B and C). Cells treated with 15d-PGJ2 and/or DPI showed no significant increase in apoptosis compared with vehicle-treated controls. Approximately 55% of cells treated with 4-HNE were apoptotic, and pretreatment with 15d-PGJ2 before exposure to 4-HNE led to a complete inhibition of apoptosis. Addition of DPI at a concentration that inhibits complex I activity by ~50% (Fig. 9A) attenuated the cytoprotective effects of 15d-PGJ2 by ~25%.



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  		Fig. 9. Inhibition of complex I attenuates the cytoprotection of 15d-PGJ2. HUVEC were treated for 16 h with varying concentrations of diphenyleneiodonium (DPI) and assayed for complex I activity (A). HUVEC were pretreated for 24 h with 2.5 µM 15d-PGJ2 and then treated for 16 h with 15 µM 4-hydroxynonenal (4-HNE) and 10 µM DPI. Apoptosis was measured with fluorescence-activated cell sorting analysis (B and C). All data are means ± SE; n = 3. *P < 0.05 compared with samples treated with both 15d-PGJ2 and 4-HNE.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Exposure to low, nontoxic levels of oxidized lipids leads to the induction of cytoprotective factors, most notably antioxidant defense systems such as MnSOD, endothelial NO synthase, and GSH, but the effects on mitochondrial function are essentially unknown (24, 26, 28, 35). In this study we have shown that exposure of endothelial cells to nontoxic levels of oxLDL and 15d-PGJ2 causes an increase in mitochondrial complex I activity. This response does not involve a change in the activity of either citrate synthase or cytochrome-c oxidase.

The precise lipids in the complex mixture in oxLDL that elicit this response are not known but can be mimicked by exposure of the cells to cyclopentenone prostaglandins, although not by lipid peroxides or aldehydes such as 4-HNE. Oxidized lipid products with the cyclopentenone electrophilic centers possessed by 15d-PGJ2 have been reported in oxLDL, and only prostaglandins that have this structural group could induce complex I activity (40). Supporting the importance of this element of the lipid structure in mediating effects on complex I, it was found that the structural analog of 15d-PGJ2, PGE2, which does not have this electrophilic center, did not change complex I activity. Interestingly, electrophilic cyclopentenones can elicit cellular responses through pathways that are both dependent and independent of the activation of the PPAR-{gamma} receptor (26). These targets include inhibition of key components of the NF-{kappa}B pathway and, as we have recently shown (26), transcriptional regulation of the rate-limiting enzyme for GSH synthesis, glutamyl cysteine ligase (39). However, exposure of HUVEC to the PPAR-{gamma} agonists troglitazone and ciglitazone and the PPAR-{alpha} agonists fenofibrate and WY-14643 resulted in only modest changes in complex I activity. Together, these data show that 15d-PGJ2 is acting independently of the PPAR-{gamma} or -{alpha} receptors to induce complex I activity.

In nonvascular cells, an association between complex I and cellular GSH has been reported in the literature. For example, in neurons it has been shown that depletion or oxidation of GSH leads to an inhibition of complex I (1, 23). In the present study we have examined this association in endothelial cells on exposure to oxLDL or 15d-PGJ2. As expected from previous studies, oxidized lipids induce the synthesis of GSH. However, this was apparently dissociated from the effects on complex I activity by a number of approaches. In the first series of experiments (Fig. 1) the exposure of the cells to the highest concentration of oxLDL resulted in a return of GSH to control levels, with no significant effect on the induction of complex I activity. In addition, direct manipulation of cellular GSH levels over a wide range of concentrations (Table 1) did not result in significant changes in either the basal or oxidized lipid-stimulated activity of complex I. Furthermore, although the addition of MnTBAP resulted in complete reversal of the capacity of 15d-PGJ2 to induce complex I activity, it had no significant effect on the induction of GSH. However, it is possible that the mitochondrial GSH pool is mediating these responses through mechanisms that are essentially independent of the cytoplasmic GSH pool. In this case we would anticipate only a weak association between complex I activity and total GSH. Recently, it has been demonstrated that S-glutathionylation of complex I can inhibit complex I activity and could conceivably contribute to the control of the enzyme in response to oxidized lipids reported here (41). Other possibilities include a direct modification of complex I subunits by the oxidized lipids. In addition, we have also shown that the induction of complex I activity by oxLDL or 15d-PGJ2 requires at least 6 h and cytoplasmic protein synthesis. Complex I consists of 45 different subunits, 38 of which are encoded for by the nuclear genome (8). Methods for analyzing changes in all of these proteins are not currently available for cells containing small amounts of mitochondria. It is possible that a nonstoichiometric synthesis of a specific nuclear encoded complex I subunit is contributing to the responses observed here. However, alternative possibilities include a dependence on complex I activity controlled by a protein not directly involved in the respiratory complex. In support of the hypothesis that transcriptional regulation may be playing a role in controlling complex I activity in response to oxidized lipids, redox-sensitive transcription factor binding sites such as AP-1, AP-2, and SP-1 have been found in the promoter regions of the NDUFA1, NDUFA5, NDUFS3, NDUFS8, NDUFV1, and NDUFV2 complex I subunit genes (13, 21, 30, 36, 42, 46). With a gene-array analysis in breast cancer cells, it was previously shown that the mRNA for subunit B18 of complex I was increased after only 2-h exposure to 15d-PGJ2 (11). Also, atherosclerotic plaques subjected to DNA microarray analysis show a 10-fold increase in the mRNA for the 51-kDa subunit of complex I (22). Because our data show no apparent change in the amounts of complex I subunits, this would seemingly exclude a transcriptional mechanism involving proteins of the respiratory complex. However, one possibility is that turnover of the proteins is a key factor and increased expression allows maintenance of the basal activity level of complex I. In this case, posttranslational modification by reactive oxygen or nitrogen species may be critical. In support of this hypothesis it has been demonstrated that exposure to 15d-PGJ2 results in the formation of ROS, derived from the mitochondria, and leads to the secondary formation of lipid oxidation products (25). Furthermore, a role for ROS/RNS in inducing complex I activity is suggested from the data in this study that indicate that compounds with antioxidant properties such as NO, probucol, and MnTBAP were able to prevent the 15d-PGJ2-dependent increase in enzyme activity. Together, these data are consistent with a role for a secondary lipid oxidation product(s) being formed on exposure of the cells to either oxLDL or 15d-PGJ2. In support of this concept, exposure of cells to SIN-1, which generates peroxynitrite from superoxide and NO and potentially causes lipid peroxidation, resulted in the induction of complex I activity. In cells, the proportion of peroxynitrite formed is lower than that in simple buffers, but sufficient amounts are formed to activate MAP kinases (27, 38).

In summary, we have demonstrated that low concentrations of oxLDL and cyclopentenones containing {alpha},{beta}-unsaturated ketones induce the activity of complex I activity but not cytochrome-c oxidase or citrate synthase in endothelial cells. The molecular mechanisms involved have not been identified but appear to include the formation of secondary oxidation products that can be inhibited by either NO or probucol and require cytoplasmic protein synthesis. This is the first demonstration of the modulation of mitochondrial protein complex activity by oxidized lipids in endothelium. We propose that this represents an adaptive, cytoprotective response of the cell to oxidative stress. In support of this hypothesis we were able to show that inhibition of complex I activity partially attenuates the cytoprotective effects of 15d-PGJ2. However, it should be noted that the inhibitor DPI is not specific to complex I, leaving open a potential contribution to oxidized lipid-dependent cytoprotection being mediated by other flavoproteins. It has recently been proposed that increased complex I activity would result in decreased formation of superoxide in the respiratory chain (32). In a multi-subunit protein of this complexity, several mechanisms could change the enzymatic activity, including posttranslational modification by protein kinases, ROS or RNS, glutathione, decreased turnover of the enzyme complex, or differential control of either the mitochondrially coded or nuclear encoded polypeptide subunits. Clearly, these aspects will require further investigation to define both the molecular mechanisms involved and the contribution to the adaptation of the endothelium to oxidative stress.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grant ES-10167 and a cardiovascular training grant (E. K. Ceaser) from NIH.


   ACKNOWLEDGMENTS
 
The authors thank Tess Hillson, Sruti Shiva, and Dr. Douglas Moellering for technical assistance and helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Darley-Usmar, Biomedical Research Bldg. II, 901 19th St. South, Birmingham, AL 35294-2180 (E-mail: darley{at}path.uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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