Published online before print September 23, 2004, doi: 10.1161/01.RES.0000146030.53089.18
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Role for Sterol Regulatory Element-Binding Protein in Activation of Endothelial Cells by Phospholipid Oxidation Products
From the Departments of Medicine (M.Y., A.L.C., J.C., A.N.D., D.K.V., P.G., J.A.B.) and Pathology (M.Y., A.L.C., D.T., J.-H.Q., M.C.F., T.H., K.M., J.A.B.), David Geffen School of Medicine at University of California Los Angeles; Bristol-Myers Squibb, Pharmaceutical Research Institute (W.-P.Y., T.K.), Princeton, NJ; Division of Biomedical Sciences (Y.L., J.Y.-J.S.), University of California, Riverside.
Correspondence to Judith A. Berliner, 13-229 CHS, 650 Charles Young South, Los Angeles, CA 90095-1732. E-mail jberliner{at}mednet.ucla.edu
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Abstract |
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Oxidized phospholipids, including oxidation products of palmitoyl-arachidonyl-phosphatidyl choline (PAPC), are mediators of inflammation in endothelial cells (ECs) and known to induce several chemokines, including interleukin-8 (IL-8). In this study, we show that oxidized PAPC (OxPAPC), which accumulates in atherosclerotic lesions, paradoxically depletes endothelial cholesterol, causing caveolin-1 internalization from the plasma membrane to the endoplasmic reticulum and Golgi, and activates sterol regulatory element-binding protein (SREBP). Cholesterol loading reversed these effects. SREBP activation resulted in increased transcription of the low-density lipoprotein receptor, a target gene of SREBP. We also provide evidence that cholesterol depletion and SREBP activation are signals for OxPAPC induction of IL-8. Cholesterol depletion by methyl-ß-cyclodextrin induced IL-8 synthesis in a dose-dependent manner. Furthermore, cholesterol loading of ECs by either the cholesterol–cyclodextrin complex or caveolin-1 overexpression inhibited OxPAPC induction of IL-8. These observations suggest that changes in cholesterol level can modulate IL-8 synthesis in ECs. The OxPAPC induction of IL-8 was mediated through the increased binding of SREBP to the IL-8 promoter region, as revealed by mobility shift assays. Overexpression of either dominant-negative SREBP cleavage-activating protein or 25-hydroxycholesterol significantly suppressed the effect of OxPAPC on IL-8 transcription. A role for SREBP activation in atherosclerosis is suggested by the observation that EC nuclei showed strong SREBP staining in human atherosclerotic lesions. The current studies suggest a novel role for endothelial cholesterol depletion and subsequent SREBP activation in inflammatory processes in which phospholipid oxidation products accumulate.
Key Words: oxidized phospholipids • interleukin-8 • endothelium • caveolae • cholesterol
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Introduction |
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Atherosclerosis is a chronic inflammatory process of vascular cells characterized by the entry of monocytes into the vessel wall at all stages of atherogenesis.1 We previously identified oxidized palmitoyl-arachidonyl-phosphatidyl choline (OxPAPC), the most active component of minimally modified low-density lipoprotein LDL (MM-LDL), as an activator of monocyte–endothelial interactions.2 This oxidized phospholipid not only accumulates in atherosclerotic lesions of mice and rabbits2 but also is increased in apoptotic cells, necrotic cells, and cells exposed to oxidative stress.3,4 The importance of these lipids in atherosclerosis is suggested by the observation that polymorphisms of enzymes that mediate the formation of oxidized lipids (ie, myeloperoxidase and lipoxygenase) and enzymes that degrade these lipids (ie, paraoxanase and platelet activating factor) alter susceptibility to atherosclerosis.2
Our previous studies have demonstrated that OxPAPC at a concentration one tenth of that present in the aorta of cholesterol-fed rabbits (
400 µg/gm tissue or 400 µg/mL of combined active lipids)5 is a strong endothelial activator. Treatment of endothelial cells (ECs) in culture with OxPAPC increases the expression of the monocyte-binding molecule CS-1 fibronectin on the apical cell surface6and synthesis of monocyte chemotactic factors (monocyte chemotactic protein 1 and interleukin-8 [IL-8]).7,8 These effects of OxPAPC were not attributable to toxicity and are reversible on its removal.7 The major identified lipids in OxPAPC responsible for IL-8 induction were 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC). Both of these phospholipids are increased in MM-LDL compared with native LDL.9
OxPAPC induction of IL-8 in ECs is transcriptionally regulated and sustained for 24 hours,8 independent of classical transcription factors nuclear factor 
B (NF-B) or activation protein 1. These findings suggest that OxPAPC induces IL-8 transcription by a previously uncharacterized signaling pathway. Our recent data identified a c-Src/STAT3 pathway, transiently activated by OxPAPC (duration of 2 to 4 hours), that mediates the early induction of IL-8.10 The transience of STAT3 activation by OxPAPC indicated that other transcription factors are responsible for the sustained increase of IL-8 transcription by OxPAPC.
Sterol regulatory element-binding proteins (SREBPs) are a family of 3 transcription factors, SREBP1a, SREBP1c, and SREBP2, that mediate cholesterol metabolism.11 SREBP is activated by SREBP cleavage-activating protein (SCAP; an endoplasmic reticulum [ER] protein) cleavage of the SREBP precursor to the active form, which translocates to the nucleus.12 Oxidized LDL (OxLDL) and OxPAPC, which contains bioactive lipids present in (including POVPC and palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine) and absent from (such as PEIPC) OxLDL,13 have been demonstrated to cause the movement of caveolin-1 from caveolae to internal membrane compartments.14 This redistribution is similar to that of methyl-ß-cyclodextrin (MBCD), an agent shown previously to deplete cells of cholesterol, leading to SREBP activation (although such activation was not examined for OxLDL).15,16 The current study presents evidence that OxPAPC and its component lipids cause cholesterol depletion, activation of SREBP, and transcription of downstream targets such as the LDL receptor (LDLR). We also demonstrate increased expression and activation of endothelial SREBP in human atherosclerosis.
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Materials and Methods |
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Reagents and Antibodies
MBCD, cholesterol (water-soluble cholesterol–cyclodextrin complexes), and 25-hydroxycholesterol (25HC) were purchased from Sigma. PAPC was purchased from Avanti Polar Lipids. OxPAPC and POVPC were prepared by procedures described previously.5 PEIPC was isolated from OxPAPC using previous methods.4 Antibodies for Western analyses were from the following sources: caveolin-1 monoclonal (BD Transduction Laboratories), calnexin polyclonal (StressGen Biotechnologies), B-COP mouse ascites fluid (Sigma), paxillin polyclonal, SREBP-1, and SREBP-2 polyclonal, and HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Western blots were developed by use of enhanced chemiluminescence (ECL) plus reagent (Amersham).
Cell Culture and Treatment
Human aortic ECs (HAECs) were isolated and maintained as described previously.7 Bovine aortic ECs (BAECs) were purchased from VEC Technologies and cultured in DMEM low-glucose media (Irvine Scientific) containing penicillin-streptomycin-glutamine and 15% FBS (Hyclone). Human cell lines (HeLas) were maintained in DMEM high-glucose media containing 10% FBS. HAECs (passages 4 to 8) and BAECs (passages 5 to 17) were used in all experiments. Human microvascular ECs (HMECs) were obtained from the Centers for Disease Control and Prevention and cultured as described previously.17 Cells were treated with OxPAPC (35 to 50 µg/mL) or MBCD (0.05 to 0.3% wt/vol) in media containing 1% FBS. For cholesterol loading, cells were either treated with cholesterol cyclodextrin complex (10 to 20 µg/mL) for 1 hour and then OxPAPC or MBCD was added for 4 hours in the presence of complex, or they were treated with complex, washed 3x, and then treated with OxPAPC or MBCD.
Western Blot Analysis
Procedures for Western blot analysis were as described previously.18 In brief, N-Acetyl-leu-leu-met at a concentration of 200 µmol/L and protease inhibitor cocktail were added to media and PBS (during cell harvest). For Western blotting, samples were separated on 4% to 12% Tris-glycine sodium dodecyl sulfate gels (Gradipore) and developed with ECL plus.
Sucrose Gradient Density Centrifugation
Cells were fractionated by the method of Parton and Hancock19 because it does not use detergent and is therefore more appropriate for lipid extraction and gas chromatography-mass spectrometry (GC-MS) analysis.
Cholesterol Measurements
Cholesterol levels were determined by extracting total lipids from medium (2.0 mL) or equal volumes of pooled cell fractions 1 to 5, which were enriched in caveolin in untreated cells and thus represented the caveolar/lipid raft fraction. Levels of total cholesterol were assessed by enzymatic colorimetric assay or by GC-MS. For analysis by GC-MS, each sample (and cholesterol standard) was spiked with 6 µg d6-(26,26,26,27,27,27)-cholesterol as an internal standard and derivatized with 80 µL of N,O,-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane (Pierce) and 20 µL of ethyl acetate at 60°C for 2 hours. Cholesterol levels were analyzed using an electron-ionization time-of-flight mass spectrometer (Micromass GCT).
Enzyme-Linked Immunosorbent Assay
IL-8 levels in HAEC supernatants were measured with use of an IL-8 ELISA kit (Quantikine Immunoassay R&D Systems) according to the protocol of the manufacturer.8
Transient Transfection and Promoter Analysis
The pcI-neo expression vector containing the full-length human caveolin-1 cDNA was a gift from Dr E. Smart (University of Kentucky).20 One human IL-8 promoter (1.4 kb) luciferase reporter construct and the NF-Bx3–responsive luciferase plasmid were obtained from Dr N. Leitinger (University of Vienna, Austria),8,21 and a second IL-8 promoter reporter construct (1.4 kb) was obtained from Dr K. Matsushima (Tokyo University, Japan).22 The expression plasmids containing active SREBP1, SREBP2, dominant-negative SCAP (ie, SCAP-C), or the human LDLR promoter reporter constructs were as described previously.23 Transient transfection experiments were performed with use of Effectene (Qiagen) or FuGENE-6 (Roche), with protocols provided by manufacturers. Twelve hours after transfection, cells were treated with the agents indicated for 12 hours. Luciferase values were normalized to ß-galactosidase activity or Renilla levels (Promega).
Quantitative Polymerase Chain Reaction
Primer sets used were: LDLR 5'-cgtgcttgtctgtcacctgcaaat-3', 5'-agaactgaggaatgcagcggttga-3'; and GAPDH 5'-cattgccctcaacgaccactttgt-3', 5'-accaccctgttgctgtagccaaat-3'. Reactions were run in triplicate on a Bio-Rad iCycler with the following thermal protocol: 94° for 5 minutes, then 40 cycles of 94° for 15 seconds, 60° for 30 seconds, and 72° for 30 seconds, ending with a slow ramp from 55° to 95° to generate melt-curve data. Comparative data were generated from sample cycle threshold values using the Pfaffl method. All primer sets showed single melt peaks and had correlations >0.99 and polymerase chain reaction (PCR) efficiencies >90%.
Electrophoretic Mobility Shift Assays
Treated cells were washed with cold PBS and lysed in the buffer containing 10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.1% Nonidet P-40 (NP-40), 0.5 mmol/L dithiothreitol (DTT), and protease inhibitors. Lysates were centrifuged at 10 000g for 10 minutes. Pellets were resuspended in buffer containing 20 mmol/L HEPES, pH 7.9, 20% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.1% NP-40, 0.5 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitors. Lysates were centrifuged at 10 000g for 10 minutes, and the supernatants were collected. Double-stranded DNA corresponding to nucleotides -140 to -120 of human IL-8 promoter containing the sterol regulatory element (SRE) was end-labeled with 32P. The radiolabeled probe was then incubated with either 5 ng recombinant SREBP-2 mature form (ie, SREBP2(N))24 or 20 µg isolated nuclear extracts for 20 minutes at 25°. The DNA–protein complexes were resolved by 5% nondenaturing acrylamide gel. The DNA–protein interactions were revealed by autoradiography.
Immunohistochemical Analysis
Sections (4-µm thick) of human carotid atherectomy specimens collected from 7 different individuals were deparaffinized and rehydrated. Immunostaining with either SREBP1 or SREBP2 antibodies (Santa Cruz) was as described previously.25
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Results |
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OxPAPC and POVPC Cause Caveolin-1 Redistribution and Change the Levels of Cholesterol in the Caveolar/Lipid Raft Fraction
We demonstrated previously that OxPAPC treatment of BAECs caused a redistribution of caveolin-1 from the caveolae/lipid raft fraction to internal membrane fractions.14 To expand these studies, the effect of OxPAPC and POVPC, as well as MCDB, on caveolin-1 distribution and cholesterol levels was examined. We were unable to test the effect of PEIPC on caveolin or cholesterol because of the difficulty of obtaining sufficient PEIPC for testing. Treatment with OxPAPC and POVPC caused caveolin-1 movement from the caveolar/lipid raft fraction to cytoplasmic fractions containing ER and Golgi markers (Figure 1). Such redistribution started as early as 2 hours after treatment with OxPAPC and continued for at least 4 hours (data not shown). The effect of OxPAPC was similar to that for MBCD26 (Figure 1B). Similar results were obtained with HAECs (Figure 1B, bottom 2 lanes).
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Because the caveolar/lipid raft fraction has been shown to be highly enriched in cholesterol compared with other cell membranes,15 we examined the effect of OxPAPC and POVPC on cholesterol levels in this fraction. Cells were exposed to medium alone, PAPC (35 µg/mL), OxPAPC (35 µg/mL), or POVPC (10 µg/mL) for these studies. As determined by enzymatic colorimetric assay or GC-MS, cholesterol levels were decreased by 30% to 40% in the caveolar/lipid raft fractions from BAECs treated with either OxPAPC or POVPC (Figure 2A). On the contrary, the level of cholesterol in the conditioned media from cells treated with OxPAPC and POVPC was increased by 50% to 60% compared with that of control medium or PAPC (Figure 2B). Similar results were observed in HAECs (data not shown). The extent of the caveolin shift relative to untreated cells (Figure 1B) differed slightly in different gradients but the percentage of caveolin in fractions 1 to 5 in cells treated with OxPAPC, POVPC, or MBCD differed from control or PAPC in all experiments. Because of this variability, it is difficult to make an exact correlation of the extent of cholesterol depletion (which was less variable) and the caveolin shift.
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Cholesterol Depletion Leads to Induction of IL-8
Next, we tested the role of cholesterol depletion in regulating IL-8 synthesis. HAECs were treated with control media, OxPAPC, POVPC, or 0.3% MBCD, and IL-8 levels were determined in the medium. MBCD increased IL-8 protein secretion into the medium to the same extent as OxPAPC and POVPC (Figure 2C). Because OxPAPC-induced IL-8 expression is transcriptionally regulated, we used IL-8 promoter reporter constructs (pIL8-Luc) to determine whether MBCD also regulated IL-8 at the transcriptional level. MBCD, at 0.3%, maximally stimulated luciferase activity (Figure 2D), but similar to OxPAPC, it did not activate NF-B (data not shown). These data suggest that endothelial cholesterol depletion, caused by OxPAPC or MBCD, increases IL-8 transcription independent of NF-B activation.
To further confirm the role for cholesterol depletion in OxPAPC-induced IL-8 expression, we tested the effect of cholesterol loading. BAECs or HAECs were treated with cholesterol–cyclodextrin complex for 30 minutes before the addition of OxPAPC to medium containing the complex. The cholesterol content of the cells was increased 2-fold by exposure to the complex, and caveolin-1 redistribution and cholesterol depletion were inhibited by exposure to the complex (Figure 3A and 3B). Cholesterol loading also abolished the OxPAPC or POVPC induction of IL-8 (Figure 3C). To be certain that cholesterol loading rather than the formation of OxPAPC–cholesterol complexes or an OxPAPC–cyclodextrin complex was responsible for the observed inhibition, ECs were exposed to cholesterol–cyclodextrin before OxPAPC treatment. Under these conditions, cholesterol was still an effective inhibitor, although slightly less, of OxPAPC- or MBCD-induced IL-8 expression (Figure 3D). In addition, caveolin-1 overexpression, which increases cholesterol content by 2-fold in BAECs,27 strongly inhibited the OxPAPC- and MBCD- but not tumor necrosis factor-
(TNF-)–activated IL-8 promoter construct (Figure 3E). These data support our hypothesis that the depletion of caveolae–cholesterol is an important signaling cue for OxPAPC-induced IL-8 expression in ECs.
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OxPAPC Activates SREBP in ECs
Because OxPAPC and MBCD caused the movement of caveolin-1 to the ER/Golgi fraction (Figure 1A and 1B), we tested whether the depletion of caveolar cholesterol by OxPAPC, as was observed previously with MBCD,16 would activate SREBP. OxPAPC treatment caused a decrease in SREBP1 precursors (125 kDa) and a concurrent increase in the mature form of SREBP1 (68 kDa) in HMECs as well as HeLa cells, another OxPAPC-responsive cell type (Figure 4A). SREBP cleavage induced by OxPAPC was observed after 1 hour of treatment and persisted at 8 hours in OxPAPC-treated HAECs (Figure 4B). However, cholesterol loading reversed the OxPAPC-activated SREBP in HeLa cells (Figure 4A) and HAECs and HMECs (data not shown). These results suggest that cholesterol depletion by OxPAPC mediates SREBP activation.
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Quantitative PCR analyses showed that OxPAPC caused a dose-dependent increase in mRNA levels of LDLR in HAECs (Figure 4C) and induced a 6-fold increase in the mRNA level of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) synthase (OxPAPC 40 µg/mL; data not shown), both known SREBP targets. PEIPC (0.3 µg/mL), the most bioactive lipid in OxPAPC, also strongly increased the level of LDLR mRNA (Figure 4C) and HMG CoA synthase mRNA (data not shown). POVPC, a less potent IL-8 inducer, only weakly increases LDLR mRNA by 2- to 3-fold (Figure 4C). In a transient transfection experiment, OxPAPC also increased LDLR promoter activation (Figure 4D), which was abolished by overexpression of a dominant-negative mutant of SCAP (SCAP-C) that inhibits movement of SREBP precursor to the Golgi.23 These studies demonstrate that cholesterol depletion by OxPAPC is sufficient to activate SREBP in ECs, which in turn increases the expression of SREBP target genes such as LDLR.
OxPAPC Induction of IL-8 Expression Requires SREBP
To determine whether SREBP activation plays a role in OxPAPC-induced IL-8 expression, we tested the effect of two SCAP inhibitors: 25HC, an agent that facilitates cholesterol translocation from plasma membrane to ER,12 and SCAP-C. One-hour pretreatment with 25HC followed by 4-hour cotreatment with OxPAPC and 25HC resulted in a significant inhibition of IL-8 protein synthesis in HAECs (Figure 5A) and IL-8 promoter activation by OxPAPC in HeLa cells (Figure 5B); 25HC did not reduce the IL-8 protein synthesis in response to phorbol myristoyl acetate (PMA) or TNF- in HAECs (data not shown). Confirming a role for SREBP in OxPAPC-induced IL-8 expression, SCAP-C was found to significantly inhibit OxPAPC activation of the IL-8 promoter construct by 60% (Figure 5C). In contrast, overexpression of the active form of SREBP1 or SREBP2 increased pIL8 promoter activity in HeLa cells (Figure 5D). To further delineate the role of SREBP2 in IL-8 induction, we performed electrophoretic mobility shift assays (EMSAs) to investigate the binding of SREBP2 to the IL-8 promoter. As shown in Figure 5E, GST-SREBP2(N) fusion protein binds to an oligonucleotide containing a consensus SRE corresponding to sequence -134 to -125 in the IL-8 promoter. Furthermore, nuclear extracts isolated from OxPAPC- or MBCD-treated cells showed increased binding to the SRE compared with those from nontreated controls. Binding was inhibited by unlabeled oligo (SC) but not by a scrambled oligo (NC). GST-SREBP2(N) and nuclear extracts only bind marginally to a second oligonucleotide (-675 to -659) that contains an SRE that differs from the consensus sequence by one base (data not shown).
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SREBP Activation in Lumenal ECs in Human Atherosclerotic Lesions
To determine the in vivo relevance of SREBP activation in ECs, we performed immunohistochemistry on human atherosclerotic lesions using antibodies that recognize active and precursor forms of SREBP. Consistent with previous data that phospholipid oxidation products accumulate in macrophage-rich areas of atherosclerotic lesions,2 we found that shoulder areas of these lesions, with large numbers of inflammatory cells, showed an overall increase in staining of SREBP-1 and SREBP-2 in nuclear and cytoplasmic regions of the lumenal endothelium (Figure 6A through 6D). Very little staining was observed in the lumenal endothelium of the fibrous cap region, which lacks inflammatory cells (data not shown). Nuclear staining, seen at a higher magnification in Figure 6D, indicates the presence of the activated form of SREBP. We also observed that SREBP nuclear staining was present in the ECs of some but not all vaso vasorum in the inflammatory regions of these atherosclerotic lesions (Figure 6E and 6F); this suggests that endothelial SREBP activation may also be involved in neovascularization. A few macrophages lacking foamy cytoplasm also showed SREBP1 and SREBP2 in the nucleus, which suggests SREBP activation (Figure 6A and 6B). Overall, these studies demonstrate SREBP activation as an important feature of vascular wall cells in atherosclerotic lesions.
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Discussion |
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Although most studies have focused on the role of SREBP in hepatic lipogenesis,11,28 several recent studies suggest that under specific stress conditions, SREBP can be activated in ECs.18,23 In this study, we demonstrate that OxPAPC treatment of ECs causes sustained activation of SREBP and induction of SREBP-targeted genes (LDLR and HMG CoA synthase); this activation was also shown to play an important role in IL-8 synthesis. The data in this and in previous studies demonstrate that SREBP activation is mediated by cholesterol depletion of cell membranes. Our studies detected cholesterol depletion in the caveolae/lipid raft fraction, which contains the majority of cell cholesterol. However, because of the continuity of cholesterol transport from the ER to the cell surface,15,29–31 we would expect other cell membranes, including the ER, where SCAP senses cholesterol, to be at least locally depleted of cholesterol.
There are several mechanisms by which OxPAPC might cause cholesterol depletion. (1) It was shown previously that cholesterol can efflux from cells to phospholipid vesicles in serum-free medium and that this efflux is enhanced by the presence of SR-B1,32 which is highly expressed in ECs (data not shown). In the current studies, OxPAPC in either medium containing 2% serum (Figure 2) or in serum-free medium (data not shown) induced cholesterol depletion of caveolae and an increase in cholesterol in the medium. It has been shown that OxPAPC, like the lipids used in previous studies, can form liposomal vesicles.33 The simplest interpretation of the movement of cholesterol from ECs to the medium after OxPAPC treatment is that cholesterol can efflux to OxPAPC or POVPC vesicles as it does to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles reported previously. (2) Another possible cause of movement of cholesterol from the ECs to the medium, in the presence of oxidized phospholipids, is the release of small membrane vesicles (that contain cholesterol). A number of laboratories have demonstrated that vesicle formation can occur in the absence of cell death and that vesicle formation is associated with activation of ECs and other cell types.34–36 We have identified several signal transduction pathways by which oxidized phospholipids rapidly activate inflammatory responses in ECs; these include activation of c-Src10 and of adenylate cyclase.37 However, inhibition of these pathways did not prevent SREBP activation (data not shown). Several other pathways activated by OxPAPC may be responsible for vesicle formation.38,39
The involvement of SREBP activation in OxPAPC induction of IL-8 transcription was demonstrated by use of inhibitors of activation (Figure 5) and expression of the active form of SREBP1 and SREBP2 in HeLa cells. We present evidence that SREBP regulates IL-8 transcription by binding to an SRE in the human IL-8 promoter. OxPAPC and MBCD increased the binding of nuclear extracts to a consensus SRE in the IL-8 promoter (Figure 5E), suggesting that SREBP increases IL-8 transcription by promoter activation.
Although hypercholesterolemia has been identified as a risk factor for atherosclerosis, and cholesterol loading of macrophages clearly contributes to atherogenesis, in vivo evidence suggests that ECs in atherosclerotic lesions are depleted of cholesterol. Studies by Kincer et al demonstrated that ECs of apolipoprotein E–null mice fed a high-fat diet have decreased caveolar–cholesterol concentration in the endothelial caveolae.40 This study also suggested that circulating OxLDL causes cholesterol depletion in ECs. Furthermore, high-density lipoprotein, which delivers cholesterol to ECs, reversed the process.41 Consistent with these in vivo findings, our data support the concept that ECs, when challenged with oxidized phospholipids present at the site of chronic inflammation, will respond with caveolar–cholesterol depletion. Our data further show that OxPAPC-induced cholesterol depletion is sufficient to activate SREBP. This mechanism appears to be specific for OxPAPC because other proinflammatory agents, including TNF-, lipopolysaccharide, and PMA, were unable to increase activation of SREPB in ECs (data not shown).
The paradoxical activation of SREBP in ECs of atherosclerotic lesions, which are bathed in high levels of lipoproteins, is a surprising finding and could accelerate lesion development by several mechanisms. (1) The induction of IL-8 by SREBP activation could cause monocyte accumulation in lesions. (2) LDLR upregulation could lead to increased LDL transport into the lesions, which would contribute to foam cell formation. (3) Increased levels of LDL processing in ECs would lead to increased oxidative stress,42 and consequent oxidation of lipids by these oxidative pathways would then predispose the arterial wall to a pathologic cycle. In addition to activation of SREBP by oxidized phospholipids, disturbed flow (a known atherogenic stimuli) can prolong SREBP activation in ECs in vitro.23 Oxidation products of phospholipids have been demonstrated to transiently activate ECs by several signal transduction pathways in addition to SREBP.10,37 A combination of these more acutely induced pathways with activation of SREBP is likely responsible for the sustained endothelial activation seen with OxPAPC.
In conclusion, our data demonstrate that OxPAPC depletes cholesterol from specialized compartments in ECs. As a consequence, SREBP is activated, causing sustained induction of IL-8 in vitro. Individual phospholipids such as PEIPC and POVPC (present in OxPAPC) also lead to SREBP activation. We also present evidence for increased endothelial SREBP expression and activation (seen as nuclear localization) in the shoulder regions (enriched in inflammatory cells) of atherosclerotic lesions, where OxPAPC accumulates. These results suggest that cholesterol depletion in ECs is an important signaling pathway in areas of inflammation where oxidized lipids accumulate. Thus, this pathway may represent a novel therapeutic target for atherosclerosis and other diseases of chronic inflammation, such as rheumatoid arthritis, containing abundant oxidized phospholipids. This study and our previous studies suggest that rapid activation of STAT3 and sustained activation of SREBP combine to regulate chemokine synthesis in ECs.
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Acknowledgments |
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This project was funded by HL07895 (M.Y.), the Giannini Family Foundation (A.L.C.), National Institutes of Health grants HL30568, HL64731 (J.A.B.), HL77448 (J.Y.-J.S.), and K12HD01400 (D.V.), and NSF CHE 0078299 (UCLA Mass Spectrometry Laboratory).
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Footnotes |
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M.Y. and A.L.C. contributed equally to this work.
Y.L., J.Y.-J.S., and J.A.B. served as cosenior authors.
Original received May 27, 2004; revision received September 10, 2004; accepted September 14, 2004.
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References |
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