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Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells
Biochimica et Biophysica Acta 1534 (2001) 139^148 www.bba-direct.com
Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells Y. Yuan, L.K. Verna, N.P. Wang, H.L. Liao, K.S. Ma, Y. Wang, Y. Zhu, M.B. Stemerman * Division of Biomedical Sciences, University of California, Riverside, CA, 92521, USA Received 11 July 2001; received in revised form 16 October 2001; accepted 18 October 2001
Abstract Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-UB (NF-UB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-UB-dependent expression was not affected. The AP-1/Ets (3891 to 3908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Cholesterol; HUVEC; Adhesion molecule
1. Introduction Hypercholesterolemia is a major risk factor for atherosclerosis. Although elevated serum cholesterol level is atherogenic, precisely how increased cholesterol a¡ects the arterial wall via endothelial cells (ECs) is not clear. An extensive amount of literature
* Corresponding author. Fax: +1-909-787-5504. E-mail address: [email protected] (M.B. Stemerman).
[1^4], including our own [5,6] suggests that increased cholesterol associated with an atherosclerotic level of low-density lipoprotein (LDL) may contribute to the atherogenic activity of LDL. Thus, we hypothesized that the cholesterol molecule itself may act as a modulator of cellular function, inducing a proin£ammatory change in ECs. Vascular ECs play a central role in regulating arterial vascular tone, in£ammation, growth, and thrombosis [7^9]. An early event and a key to the development of atherosclerotic plaque is increased EC expression of adhesion molecules. Intercellular
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adhesion molecule-1 (ICAM-1) expression can be greatly increased by a variety of stimuli such as bacterial lipopolysaccharide (LPS), phorbol esters, proin£ammatory cytokines (TNF-K, IL-1 and IFNQ) [10], and LDL exposure [11]. LDL can increase monocyte adhesion and induce ICAM-1 expression in human umbilical vein ECs (HUVECs) [11]. LDL also activates the nuclear transcription factor activator protein-1 (AP-1), which is demonstrated by increased c-Jun mRNA and AP-1 binding [6]. Composed of cholesterol, apolipoproteins and other lipids, LDL mainly serves as a carrier for tissue delivery of cholesterol. Modi¢cation of cholesterol content in plasma membrane appears su¤cient to alter membrane £uidity and cellular functions, including carrier-mediated transport, the properties of certain membrane-bound proteins and cell growth [12,13]. LDL increases cellular cholesterol content and cholesterol:phospholipid ratios of EC membranes, resulting in a reduction in the relative EC plasma membrane £uidity [5]. In the present study, we investigated the role of cholesterol in EC activation and the underlying mechanism. In particular, we examined the e¡ects of cholesterol on ICAM-1 regulation and the upstream signaling events responsible. Using cholesterol-enriched liposomes, we demonstrated that cholesterol transcriptionally upregulates ICAM-1 expression and the AP-1 pathway, which are important for converting the endothelium to a proin£ammatory state. This is the ¢rst report that shows direct evidence of the role cholesterol can play in EC activation. 2. Materials and methods 2.1. Cell culture HUVEC isolation and maintenance were previously described [14]. Brie£y, cells were extracted by collagenase digestion and cultured on plates coated with collagen. Cells were maintained in medium 199 supplemented with 20 mmol/l HEPES (pH 7.4), 20% fetal bovine serum, 5 ng/ml of recombinant human ¢broblast growth factor, antibiotics/micotics and 90 Wg/ml of heparin (HUVEC medium). All experiments were performed with cells from passages 2 or 3.
2.2. Liposome preparation Liposomes were prepared by mixing cholesterol with phosphatidylserine (PS) as described [15,16]. Brie£y, cholesterol and PS were dissolved in chloroform:methanol (2:1) at a prescribed molar ratio, mixed together and dried under a gentle stream of nitrogen gas. The dried membrane was then hydrolyzed in PBS followed by sonication and extrusion through two stacked 100-nm polycarbonate ¢lters (Nuclepore) using a thermostat extruder (Lipex Biomembranes). Cholesterol-enriched liposomes (cholesterol:PS ratio of 1:1, cholesterol concentration 5 mg/ ml) were used unless otherwise indicated. The cholesterol-liposome preparation contained less than 0.0005 units of endotoxin per mg of cholesterol as determined by the chromogenic Limulus test (Bio Whittaker) as described previously [6]. 2.3. Cellular cholesterol and phospholipid measurement Cholesterol-enriched liposome-treated ECs were washed three times each with 10 ml of phosphatebu¡ered saline (PBS) before lipid extraction. Cellular lipids were extracted by the method of Folch [17]. Aliquots of the Folch extract were dried, resuspended in 0.1% Triton X-100. Cholesterol was measured with the cholesterol-oxidase method (Sigma). Quanti¢cation of phospholipid was determined by the method of Bartlett [18]. 2.4. Fluorescence-linked immunoassay of ICAM-1 Surface ICAM-1 protein was determined with use of an immuno£uorescent assay based on protocols established by Pober et al. [19]. Brie£y, after cholesterol exposure, HUVECs were trypsinized and washed. A primary antibody against ICAM-1 (Serotec, Oxford, UK) was added to the cells and incubated for 1 h on ice. The cells bound with primary antibody were then washed and a FITC-labeled secondary antibody was added and incubated for another hour on ice. After being washed, the cell suspension was transferred to a Fluoricon assay plate precoated with a 1:20 dilution of 0.84-Wm polystyrene beads (5% wt/vol; IDEXX). After sample ¢ltration, £uorescence was measured (excitation, 485 nm;
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emission, 535 nm) [11]. Cells treated with Phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) for 24 h were used as a positive control. Results were expressed as the level of £uorescence detected for ICAM-1 in cholesterol-exposed cells versus that in control HUVECs. 2.5. Northern analysis Total RNA was isolated using Trizol reagent (Gibco BRL, Grand Island, NY, USA) and subjected to Northern analysis for ICAM-1 expression. The ICAM-1 cDNA probes were generated as described [14]. Bindings of the probe to corresponding message were detected by autoradiography. Membranes were subsequently stripped and rehybridized with 32 P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. 2.6. Electrophoretic mobility shift assay (EMSA) HUVECs were cultured to con£uence in 100-mm dishes and then exposed to cholesterol for di¡erent periods of time. Nuclear proteins were extracted as previously described [6]. The consensus sequences for AP-1, nuclear factor-UB (NF-UB) or ICAM-1-speci¢c AP-1, NF-UB-like sequences were end-labeled with [Q-32 P]ATP using T4 DNA kinase. Binding of the labeled oligonucleotides to their corresponding factors was performed as previously described [6]. To test the speci¢city of binding, a 100 times molar excess of unlabeled competing oligonucleotide was used for competition experiments.
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24 h with or without cholesterol exposure. A parallel transfected group was treated with 50 ng/ml PMA for 16 h before the samples were collected and used as a positive control. Luciferase activity was measured and normalized against L-galactosidase. 2.8. Statistics All values are expressed as the mean þ S.D. Statistical signi¢cance of the data was evaluated by the Student's t-test. A P value of 0.05 or less was considered signi¢cant. All the experiments were repeated at least three times. 3. Results 3.1. Cholesterol increases cellular cholesterol:phospholipid ratio in HUVECs To evaluate the e¡ects of cholesterol-enriched liposome exposure on the lipid composition of HUVECs, we measured the cellular cholesterol:phospholipid ratio in whole cell preparations. Cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) were used to treat HUVECs for
2.7. Plasmids and transfection The promoter reporter constructs for pAP1-Luc (7xAP-1), pNFUB-Luc (5xNF-UB) were purchased from Stratagene (La Jolla, CA, USA). To construct the ICAM-1 promoter reporter, a 0.98-kb fragment containing the 5P-£anking region [20] of human ICAM-1 gene was subcloned into pGL-3 (unpublished). All plasmids were puri¢ed using a Qiagen kit. HUVECs were seeded on 6-well plates and were 50^80% con£uent prior to transfection. Plasmids were transfected using Targefect F-1 (Targeting Systems). The post-transfected cells were incubated in EC medium for 16^18 h, and then incubated for
Fig. 1. E¡ect of cholesterol-enriched liposomes on cellular cholesterol:phospholipid ratio in HUVECs. HUVECs were exposed to cholesterol-enriched liposomes (cholesterol:PS molar ratio 1:1, cholesterol concentration 5 mg/dl) for times as indicated. HUVECs without cholesterol exposure were used as control (Ctrl). Cholesterol-enriched liposome-treated ECs were washed three times each with 10 ml PBS before lipid extraction. Cellular lipids were extracted. Cholesterol was measured with a cholesterol-oxidase kit. Quanti¢cation of phospholipid was determined by the method of Bartlett [18]. The molar ratio of cholesterol:phospholipid was calculated as indicated. Data are presented as mean þ S.D. of six separate experiments performed in triplicate.
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di¡erent periods of time as indicated. As shown in Fig. 1, the cholesterol:phospholipid ratio of the cholesterol-loaded HUVECs was signi¢cantly greater compared to that for controls starting at 1 h after treatment (0.38 vs. 0.27, P 6 0.05). The cholesterol: phospholipid ratio remained elevated up to 48 h, which was the length of the test. This result suggests indirectly that cholesterol was loaded into the cells over time. 3.2. Cholesterol increases ICAM-1 surface expression in HUVECs To determine whether cholesterol had an e¡ect on ICAM-1 expression, we examined ICAM-1 protein levels in response to cholesterol exposure. Cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) were used to treat HUVECs for 6 h, 24 h and 48 h. Cells were then analyzed for the presence of ICAM-1 expression at the EC surface. Result of £uorescence analysis revealed increased ICAM-1 surface expression beginning at 24 h, which reached 2.2-fold induction at 48 h. PMA, a positive control, increased ICAM-1 surface expression more than three-fold at 24 h (Fig. 2).
Fig. 2. E¡ect of cholesterol on ICAM-1 surface expression in HUVECs. HUVECs were incubated with cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) for 6, 24 and 48 h. ICAM-1 expression was measured with use of £uorescence-linked immunoassay. PMA (50 ng/ml) treatment of 24 h was used as a positive control. Results are expressed as amount of induction compared to that in HUVECs treated with EC medium only at each time point. Data are presented as the mean þ S.D. of relative £uorescence intensities of three independent experiments, each performed in triplicate.
Fig. 3. E¡ect of cholesterol on ICAM-1 mRNA levels. (A) HUVECs were exposed to cholesterol-enriched liposomes for times indicated. HUVECs without cholesterol treatment were used as controls. Total RNA was isolated and hybridized to an ICAM1 cDNA probe. Expression of GAPDH was used as an internal control. (B) HUVECs were exposed to liposomes of di¡erent cholesterol:PS ratios with increasing cholesterol concentrations or di¡erent dosages (5 mg/dl or 10 mg/dl) of cholesterol in the same ratio (cholesterol:PS, 1:1) for 48 h. Northern analyses were done as described above.
3.3. Cholesterol increases ICAM-1 mRNA in HUVECs To explore the mechanism by which cholesterol upregulates ICAM-1 expression, we examined the steady state mRNA levels of ICAM-1. As seen in Fig. 3A, cholesterol causes ICAM-1 induction in a time-dependent fashion. Cholesterol-induced ICAM1 gene expression began at 24 h and remained elevated at 96 h, which is the length of the test. To further characterize the e¡ect of cholesterol on ICAM-1 expression, we prepared liposomes of the following cholesterol:PS ratios: 0:1, 0.25:1, 0.5:1, 1:1, 1.5:1. The ¢nal cholesterol concentration for treatment ranged from 0 to 7.5 mg/dl and the concentrations of PS were kept constant (10 mg/dl). Total RNA was harvested after 48 h of treatment. Fig. 3B shows that increased cholesterol content of liposomes could e¡ectively increase ICAM-1 mRNA expression. If the cholesterol:PS ratio was kept at 1:1, the increase in cholesterol correspondingly increased ICAM-1 mRNA (Fig. 3B).
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Fig. 4. Cholesterol transcriptionally activates ICAM-1 in HUVECs. (A) ICAM-1 mRNA decay in presence of actinomycin D. Con£uent HUVECs were treated with TNF-K (2 ng/ml) for 2 h followed by actinomycin D (5 mg/ml) with or without cholesterol, and total cellular RNA was isolated at 0, 1, 2, 4, and 6 h after treatment. ICAM-1 and GAPDH mRNA was detected by Northern blotting followed by densitometric measurement of the corresponding signal. The relative density of ICAM-1 was normalized with GAPDH. Results represent three separate experiments. (B) E¡ect of cholesterol on ICAM-1 promoter. Subcon£uent HUVECs were transfected with PGL3-0.98 plus pCMV-L-gal and then incubated for 24 h in EC medium with no supplementation (Ctrl) or with cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/ml). PMA (50 ng/ml) overnight treatment was used as positive control. Samples were collected and assayed for luciferase expression. The results were normalized against L-galactosidase. Data are presented as the mean þ S.D. of relative luciferase activities in ¢ve independent experiments, each performed in triplicate.
3.5. Cholesterol increases AP-1 binding activity
3.4. Cholesterol transcriptionally upregulates ICAM-1 To further explore the mechanism by which cholesterol upregulates ICAM-1 mRNA, we examined the upstream regulatory region of the ICAM-1 gene by transfecting HUVECs with an ICAM-1 promoterdriven reporter construct. Fig. 4B shows that cholesterol increases the promoter activity of the PGL30.98 construct by 2.65 þ 0.18-fold (P 6 0.05). To evaluate the ICAM-1 mRNA half-life in HUVECs after cholesterol treatment, we measured the time course of ICAM-1 decay in HUVECs after arrest of new transcription with actinomycin D (5 Wg/ml). HUVECs were pretreated with TNF-K (2 ng/ml) for 2 h to induce ICAM-1 expression. Total RNA was extracted after 0, 1, 2, 4, and 6 h of actinomycin D treatment and hybridized with probes for ICAM-1 or GAPDH. The half-life of ICAM-1 mRNA was 7 h with and without cholesterol exposure (Fig. 4B), which suggests that ICAM-1 mRNA stability was not a¡ected by cholesterol.
Con£uent HUVECs were exposed to cholesterolenriched liposomes for 2 h, 6 h and 12 h. The EMSA result in Fig. 5A shows that cholesterol increases AP1 binding as early as 2 h after exposure. The increased AP-1 binding activity remained high at all the time points examined. In contrast, NF-UB binding, which was markedly activated by PMA, was virtually absent in cholesterol-exposed HUVECs (Fig. 5B). As shown in Fig. 5C, only cholesterol-enriched liposomes increased AP-1 binding. Decreasing the cholesterol:PS ratio to 0.25:1 diminished this effect. In addition, cholesterol increased AP-1 binding in a dose-dependent fashion beginning at a cholesterol concentration of 2.5 mg/dl (Fig. 5D). 3.6. Cholesterol functionally increases AP-1-dependent protein expression Cholesterol increased AP-1 but not NF-UB binding as demonstrated by gel shift assays. To further
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Fig. 5. Cholesterol e¡ects on AP-1 and NF-UB binding by EMSA. HUVECs were exposed to cholesterol-enriched liposomes, and nuclear proteins were extracted. (A) and (B) Cholesterol:PS molar ratio 1:1, cholesterol concentration 5 mg/dl. (C) Cholesterol:PS with varying ratios. (D) Cholesterol with varying concentration for 6 h. EMSA was performed with use of an oligonucleotide of consensus AP-1 or NF-UB binding sequences as probe. Results are representative of three to four separate experiments.
determine whether cholesterol could speci¢cally activate an AP-1-driven promoter, we examined the effect of cholesterol by transfecting HUVECs with the pAP1-Luc construct or pNFUB-Luc construct. As shown in Fig. 6, cholesterol increased the promoter activities driven only by the AP-1 motif (P 6 0.05) but not those driven by the NF-UB motif. PMA, on the other hand, signi¢cantly activated both AP1- and NF-UB-dependent reporter luciferase activities (P 6 0.01). Thus, cholesterol speci¢cally upregulates genes driven by the AP-1 motif and has no e¡ect on the NF-UB-dependent reporter, which is highly responsive to PMA. 3.7. AP-1 is involved in cholesterol-induced upregulation of ICAM-1 The 5P-£anking region of the ICAM-1 gene con-
Fig. 6. E¡ect of cholesterol on expression of AP-1-driven or NF-UB-driven reporter gene. Subcon£uent HUVECs were cotransfected with pCMV-L-gal plus pAP1-Luc or pNFUB-Luc. After cholesterol-enriched liposomes or PMA (50 ng/ml) treatment, samples were collected and assayed for luciferase expression. The results were normalized against L-galactosidase. Data are presented as the mean þ S.D. of relative luciferase activities in ¢ve independent experiments.
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Fig. 7. E¡ect of cholesterol on ICAM-1 promoter region elements binding. HUVECs were pretreated with cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) for 2, 6, 12 h followed by nuclear extract isolation. PMA (50 ng/ ml) treatment was applied as a positive control. EMSA was performed with use of the oligonucleotide of ICAM-1 promoter region AP-1/Ets element (left panel) or NF-UB fragment (right panel) as probes. Results represent three separate experiments.
tains three putative AP-1-like sites and variant NFUB elements [21^24]. We used three synthesized oligo-DNA fragments containing the sequences of the three variant AP-1-like elements (AP1/TRE, 3321; AP-1/Ets, 3940; AP1, 31290) located in the 5P-untranslated region (UTR) of ICAM-1 gene as probes for EMSA [25]. Of the three AP-1-like elements, only the AP-1/Ets site responded to cholesterol exposure (Fig. 7, left panel). However, cholesterol did not increase binding activity of the ICAM-1 promoter region NF-UB site (3223), but did increase binding activity in the PMA-treated sample (Fig. 7, right panel). 4. Discussion This study is aimed at examining the hypothesis that cholesterol enrichment alone may induce EC activation, manifested by the regulation of adhesion molecules such as ICAM-1 expression in HUVECs. Cholesterol-enriched liposomes have been shown to transfer cholesterol to the cell membranes [26^28]. In this report, we utilized cholesterol-enriched liposomes as a cholesterol-loading system, to investigate the e¡ects of cholesterol enrichment on EC adhesion molecules, especially ICAM-1 regulation and upstream signaling. We demonstrated that (1) cholesterol-enriched liposome exposure increases the HUVEC cholesterol:phospholipid ratio, (2) cholesterol
transcriptionally upregulates ICAM-1, and (3) AP-1 and not NF-UB appears to play an important role in cholesterol-induced EC activation. This study shows, for the ¢rst time, that cholesterol, as a cellular function modulator, can activate ECs, likely through an AP-1-speci¢c pathway, leading to proin£ammatory and hence, potentially, pro-atherogenic changes. Cholesterol is an essential constituent of cells. Regulation of synthesis, in£ux and e¥ux keeps cellular cholesterol levels carefully controlled. Alterations in cellular cholesterol content have been shown to a¡ect membrane permeability, endocytosis, glucose transport, LDL binding, and cell cycle control [8,9, 29,30]. However, the role of cholesterol in activating ECs to a proin£ammatory state has not been studied. An objective in understanding the pathogenesis of hypercholesterolemia in atherogenesis is to determine how cholesterol enrichment of the endothelium affects EC function. The current study suggests that cholesterol can induce EC activation, likely through an AP-1 pathway, and can lead to ICAM-1 induction. Numerous articles, including our own, have demonstrated that LDL, a risk factor for atherosclerosis, is an EC activator [5,6,31]. LDL is a major carrier of cholesterol in vivo. Our previous study showed that LDL can activate ICAM-1 mainly through an AP-1mediated pathway [25]. One of the objectives in understanding LDL-induced atherogenesis is to determine the role of di¡erent moieties of the LDL
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particle and the mechanism by which they alter endothelial function. The present report extends our previous studies by focusing on cholesterol as a major stimulator. Striking similarities were observed in cholesterol- and LDL-induced EC activation. First, both can upregulate ICAM-1 expression. Second, both activate AP-1 but not NF-UB. Lastly, both enrich the EC cellular membrane with cholesterol. Our ¢nding indicates that many important aspects related to the role of LDL in atherosclerosis are directly linked to the accumulation of cholesterol in ECs. Adhesion molecules play a key role in the recruitment and extravasation of circulating leukocytes at the site of in£ammation [32]. ICAM-1 upregulation has been observed in lesional or lesion-prone areas of human atherosclerosis and hypercholesterolemic animals [33,34]. In ECs, ICAM-1 is upregulated in response to a wide variety of stimuli including proin£ammatory cytokines, LPS, PMA and H2 O2 . These stimuli increase ICAM-1 expression primarily through activation of ICAM-1 gene transcription [35]. Thus far, more than 4 kb of the 5P-£anking DNA of the human ICAM-1 gene has been cloned and sequenced [22,23]. Analysis of the 1.3-kb human ICAM-1 promoter region reveals multiple binding motifs for various transcription activators including three putative AP-1-like sites, variant NF-UB elements [21], AP-2, Ets, STATs and SP-1 sites, which suggests complex transcriptional regulation [22^24]. The variant NF-UB site in the promoter is responsive to cytokines and LPS and has been the target of several studies [36]. Sequence analysis indicates that AP-1 binding sites are recurrent elements in the promoters of many proin£ammatory genes such as ICAM-1, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). In the present study, we found that among the three AP-1-like elements in the ICAM-1 promoter region, the AP-1/Ets (3891 to 3908) site is most responsive to cholesterol exposure. However, we did not observe increased binding of the NF-UB-like element, which is consistent with our previous ¢nding in LDL-induced ICAM-1 transcriptional activation [6]. Both AP-1 and NF-UB transcription factors have been reported to be involved in the regulation of ICAM-1 [21,24,36^38]. In ECs, deregulation of AP1 can be seen in response to a variety of pathophysiologic stimuli such as cytokines [39], bacterial en-
dotoxin, antioxidants [40], hypoxia [41], and £uid shear stress [42]. Particularly, LDL induces a sustained activation of AP-1 in ECs [6,43,44]. Although NF-UB has been shown to play an important role in EC activation [45^47], increasing evidence indicates the importance of AP-1 in endothelial homeostasis and dysfunction. We recently demonstrated the induction of ICAM-1 and MCP-1 independent of NF-UB activation [25]. Overexpression of AP-1 components alone directly causes ICAM-1 induction in ECs [25], which con¢rms that AP-1 itself is su¤cient to cause phenotypic activation and induction of ICAM-1 in ECs. In contrast to most proin£ammatory cytokines, growth factors and bacterial endotoxin, cholesterol has no e¡ect on NF-UB. Interestingly, this result is consistent with our previous ¢nding that LDL activates AP-1 but not NF-UB in ECs [6]. Further, AP-1- and NF-UB-driven promoter transfections revealed that AP-1 activation is mainly responsible for cholesterol-induced EC activation, and NFUB appears not to be involved in this process. Cholesterol appears to activate ICAM-1 predominantly through an AP-1-mediated mechanism. Similarly, a stimulus-speci¢c NF-UB-independent mechanism was reported in the induction of the IL-8 gene [48]. Cholesterol appears to be an endothelial agonist distinct from other cell stimulators such as cytokines, endotoxin, oxidized LDL, and phorbol esters by activating ECs predominantly through transcription factor AP-1. Thus, enrichment of cholesterol, as a chronic vascular agonist, may perturb the EC phenotype through a mechanism that is distinct from an acutely activated pathway. A correlate to this is that atherosclerotic plaque formation is a chronic process, in keeping with the present results. The mechanistic basis for the e¡ect of cholesterol enrichment on cellular function modulation can be interpreted from the actions of cholesterol on the membrane lipid bilayer. Cholesterol is non-randomly distributed between lea£ets of the membrane bilayer, and the cholesterol distribution in membranes is highly dynamic. This o¡ers numerous possibilities for explaining the in£uence of cholesterol on membrane-embedded proteins and subsequent signal transduction. Cholesterol e¡ects can be caused by its modi¢cation of cellular membrane £uidity, which has been seen in red blood cells, hepatocytes, platelet, epithelial cells and SMCs [29]. Prolonged incuba-
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tion of ECs with native LDL is associated with marked reduction in relative EC plasma membrane £uidity [5]. Likewise, exposure of ECs to cholesterol may decrease cell membrane £uidity, perturb the cell surface receptors, and thereby a¡ect intracellular signaling. In conclusion, we demonstrate, for the ¢rst time, that cholesterol enrichment of ECs activates the AP1 signaling pathway, resulting in an increase in ICAM-1 expression. This leads to the induction of endothelial adhesion molecule upregulation and thus EC activation. This ¢nding is consistent with the hypothesis that cholesterol enrichment of ECs mediates initiation of atherogenesis following the development of in vivo serum hypercholesterolemia. To our knowledge, this is the ¢rst evidence of the crucial role of the cholesterol molecule in EC activation. This ¢nding may contribute to our understanding of the mechanisms of hypercholesterol-induced EC dysfunction.
[7] [8] [9] [10]
[11]
[12] [13]
[14]
[15]
Acknowledgements This study was supported by the NIH grant HL43023 (to M.B.S.). We thank Dr. D. Johnson for his thoughtful critiques of the manuscript. We thank Dr. J.A. Thompson for providing recombinant human ¢broblast growth factor.
[16]
[17]
[18] [19]
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Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells Y. Yuan, L.K. Verna, N.P. Wang, H.L. Liao, K.S. Ma, Y. Wang, Y. Zhu, M.B. Stemerman * Division of Biomedical Sciences, University of California, Riverside, CA, 92521, USA Received 11 July 2001; received in revised form 16 October 2001; accepted 18 October 2001
Abstract Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-UB (NF-UB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-UB-dependent expression was not affected. The AP-1/Ets (3891 to 3908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Cholesterol; HUVEC; Adhesion molecule
1. Introduction Hypercholesterolemia is a major risk factor for atherosclerosis. Although elevated serum cholesterol level is atherogenic, precisely how increased cholesterol a¡ects the arterial wall via endothelial cells (ECs) is not clear. An extensive amount of literature
* Corresponding author. Fax: +1-909-787-5504. E-mail address: [email protected] (M.B. Stemerman).
[1^4], including our own [5,6] suggests that increased cholesterol associated with an atherosclerotic level of low-density lipoprotein (LDL) may contribute to the atherogenic activity of LDL. Thus, we hypothesized that the cholesterol molecule itself may act as a modulator of cellular function, inducing a proin£ammatory change in ECs. Vascular ECs play a central role in regulating arterial vascular tone, in£ammation, growth, and thrombosis [7^9]. An early event and a key to the development of atherosclerotic plaque is increased EC expression of adhesion molecules. Intercellular
1388-1981 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 1 ) 0 0 1 8 8 - 3
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adhesion molecule-1 (ICAM-1) expression can be greatly increased by a variety of stimuli such as bacterial lipopolysaccharide (LPS), phorbol esters, proin£ammatory cytokines (TNF-K, IL-1 and IFNQ) [10], and LDL exposure [11]. LDL can increase monocyte adhesion and induce ICAM-1 expression in human umbilical vein ECs (HUVECs) [11]. LDL also activates the nuclear transcription factor activator protein-1 (AP-1), which is demonstrated by increased c-Jun mRNA and AP-1 binding [6]. Composed of cholesterol, apolipoproteins and other lipids, LDL mainly serves as a carrier for tissue delivery of cholesterol. Modi¢cation of cholesterol content in plasma membrane appears su¤cient to alter membrane £uidity and cellular functions, including carrier-mediated transport, the properties of certain membrane-bound proteins and cell growth [12,13]. LDL increases cellular cholesterol content and cholesterol:phospholipid ratios of EC membranes, resulting in a reduction in the relative EC plasma membrane £uidity [5]. In the present study, we investigated the role of cholesterol in EC activation and the underlying mechanism. In particular, we examined the e¡ects of cholesterol on ICAM-1 regulation and the upstream signaling events responsible. Using cholesterol-enriched liposomes, we demonstrated that cholesterol transcriptionally upregulates ICAM-1 expression and the AP-1 pathway, which are important for converting the endothelium to a proin£ammatory state. This is the ¢rst report that shows direct evidence of the role cholesterol can play in EC activation. 2. Materials and methods 2.1. Cell culture HUVEC isolation and maintenance were previously described [14]. Brie£y, cells were extracted by collagenase digestion and cultured on plates coated with collagen. Cells were maintained in medium 199 supplemented with 20 mmol/l HEPES (pH 7.4), 20% fetal bovine serum, 5 ng/ml of recombinant human ¢broblast growth factor, antibiotics/micotics and 90 Wg/ml of heparin (HUVEC medium). All experiments were performed with cells from passages 2 or 3.
2.2. Liposome preparation Liposomes were prepared by mixing cholesterol with phosphatidylserine (PS) as described [15,16]. Brie£y, cholesterol and PS were dissolved in chloroform:methanol (2:1) at a prescribed molar ratio, mixed together and dried under a gentle stream of nitrogen gas. The dried membrane was then hydrolyzed in PBS followed by sonication and extrusion through two stacked 100-nm polycarbonate ¢lters (Nuclepore) using a thermostat extruder (Lipex Biomembranes). Cholesterol-enriched liposomes (cholesterol:PS ratio of 1:1, cholesterol concentration 5 mg/ ml) were used unless otherwise indicated. The cholesterol-liposome preparation contained less than 0.0005 units of endotoxin per mg of cholesterol as determined by the chromogenic Limulus test (Bio Whittaker) as described previously [6]. 2.3. Cellular cholesterol and phospholipid measurement Cholesterol-enriched liposome-treated ECs were washed three times each with 10 ml of phosphatebu¡ered saline (PBS) before lipid extraction. Cellular lipids were extracted by the method of Folch [17]. Aliquots of the Folch extract were dried, resuspended in 0.1% Triton X-100. Cholesterol was measured with the cholesterol-oxidase method (Sigma). Quanti¢cation of phospholipid was determined by the method of Bartlett [18]. 2.4. Fluorescence-linked immunoassay of ICAM-1 Surface ICAM-1 protein was determined with use of an immuno£uorescent assay based on protocols established by Pober et al. [19]. Brie£y, after cholesterol exposure, HUVECs were trypsinized and washed. A primary antibody against ICAM-1 (Serotec, Oxford, UK) was added to the cells and incubated for 1 h on ice. The cells bound with primary antibody were then washed and a FITC-labeled secondary antibody was added and incubated for another hour on ice. After being washed, the cell suspension was transferred to a Fluoricon assay plate precoated with a 1:20 dilution of 0.84-Wm polystyrene beads (5% wt/vol; IDEXX). After sample ¢ltration, £uorescence was measured (excitation, 485 nm;
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emission, 535 nm) [11]. Cells treated with Phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) for 24 h were used as a positive control. Results were expressed as the level of £uorescence detected for ICAM-1 in cholesterol-exposed cells versus that in control HUVECs. 2.5. Northern analysis Total RNA was isolated using Trizol reagent (Gibco BRL, Grand Island, NY, USA) and subjected to Northern analysis for ICAM-1 expression. The ICAM-1 cDNA probes were generated as described [14]. Bindings of the probe to corresponding message were detected by autoradiography. Membranes were subsequently stripped and rehybridized with 32 P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. 2.6. Electrophoretic mobility shift assay (EMSA) HUVECs were cultured to con£uence in 100-mm dishes and then exposed to cholesterol for di¡erent periods of time. Nuclear proteins were extracted as previously described [6]. The consensus sequences for AP-1, nuclear factor-UB (NF-UB) or ICAM-1-speci¢c AP-1, NF-UB-like sequences were end-labeled with [Q-32 P]ATP using T4 DNA kinase. Binding of the labeled oligonucleotides to their corresponding factors was performed as previously described [6]. To test the speci¢city of binding, a 100 times molar excess of unlabeled competing oligonucleotide was used for competition experiments.
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24 h with or without cholesterol exposure. A parallel transfected group was treated with 50 ng/ml PMA for 16 h before the samples were collected and used as a positive control. Luciferase activity was measured and normalized against L-galactosidase. 2.8. Statistics All values are expressed as the mean þ S.D. Statistical signi¢cance of the data was evaluated by the Student's t-test. A P value of 0.05 or less was considered signi¢cant. All the experiments were repeated at least three times. 3. Results 3.1. Cholesterol increases cellular cholesterol:phospholipid ratio in HUVECs To evaluate the e¡ects of cholesterol-enriched liposome exposure on the lipid composition of HUVECs, we measured the cellular cholesterol:phospholipid ratio in whole cell preparations. Cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) were used to treat HUVECs for
2.7. Plasmids and transfection The promoter reporter constructs for pAP1-Luc (7xAP-1), pNFUB-Luc (5xNF-UB) were purchased from Stratagene (La Jolla, CA, USA). To construct the ICAM-1 promoter reporter, a 0.98-kb fragment containing the 5P-£anking region [20] of human ICAM-1 gene was subcloned into pGL-3 (unpublished). All plasmids were puri¢ed using a Qiagen kit. HUVECs were seeded on 6-well plates and were 50^80% con£uent prior to transfection. Plasmids were transfected using Targefect F-1 (Targeting Systems). The post-transfected cells were incubated in EC medium for 16^18 h, and then incubated for
Fig. 1. E¡ect of cholesterol-enriched liposomes on cellular cholesterol:phospholipid ratio in HUVECs. HUVECs were exposed to cholesterol-enriched liposomes (cholesterol:PS molar ratio 1:1, cholesterol concentration 5 mg/dl) for times as indicated. HUVECs without cholesterol exposure were used as control (Ctrl). Cholesterol-enriched liposome-treated ECs were washed three times each with 10 ml PBS before lipid extraction. Cellular lipids were extracted. Cholesterol was measured with a cholesterol-oxidase kit. Quanti¢cation of phospholipid was determined by the method of Bartlett [18]. The molar ratio of cholesterol:phospholipid was calculated as indicated. Data are presented as mean þ S.D. of six separate experiments performed in triplicate.
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di¡erent periods of time as indicated. As shown in Fig. 1, the cholesterol:phospholipid ratio of the cholesterol-loaded HUVECs was signi¢cantly greater compared to that for controls starting at 1 h after treatment (0.38 vs. 0.27, P 6 0.05). The cholesterol: phospholipid ratio remained elevated up to 48 h, which was the length of the test. This result suggests indirectly that cholesterol was loaded into the cells over time. 3.2. Cholesterol increases ICAM-1 surface expression in HUVECs To determine whether cholesterol had an e¡ect on ICAM-1 expression, we examined ICAM-1 protein levels in response to cholesterol exposure. Cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) were used to treat HUVECs for 6 h, 24 h and 48 h. Cells were then analyzed for the presence of ICAM-1 expression at the EC surface. Result of £uorescence analysis revealed increased ICAM-1 surface expression beginning at 24 h, which reached 2.2-fold induction at 48 h. PMA, a positive control, increased ICAM-1 surface expression more than three-fold at 24 h (Fig. 2).
Fig. 2. E¡ect of cholesterol on ICAM-1 surface expression in HUVECs. HUVECs were incubated with cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) for 6, 24 and 48 h. ICAM-1 expression was measured with use of £uorescence-linked immunoassay. PMA (50 ng/ml) treatment of 24 h was used as a positive control. Results are expressed as amount of induction compared to that in HUVECs treated with EC medium only at each time point. Data are presented as the mean þ S.D. of relative £uorescence intensities of three independent experiments, each performed in triplicate.
Fig. 3. E¡ect of cholesterol on ICAM-1 mRNA levels. (A) HUVECs were exposed to cholesterol-enriched liposomes for times indicated. HUVECs without cholesterol treatment were used as controls. Total RNA was isolated and hybridized to an ICAM1 cDNA probe. Expression of GAPDH was used as an internal control. (B) HUVECs were exposed to liposomes of di¡erent cholesterol:PS ratios with increasing cholesterol concentrations or di¡erent dosages (5 mg/dl or 10 mg/dl) of cholesterol in the same ratio (cholesterol:PS, 1:1) for 48 h. Northern analyses were done as described above.
3.3. Cholesterol increases ICAM-1 mRNA in HUVECs To explore the mechanism by which cholesterol upregulates ICAM-1 expression, we examined the steady state mRNA levels of ICAM-1. As seen in Fig. 3A, cholesterol causes ICAM-1 induction in a time-dependent fashion. Cholesterol-induced ICAM1 gene expression began at 24 h and remained elevated at 96 h, which is the length of the test. To further characterize the e¡ect of cholesterol on ICAM-1 expression, we prepared liposomes of the following cholesterol:PS ratios: 0:1, 0.25:1, 0.5:1, 1:1, 1.5:1. The ¢nal cholesterol concentration for treatment ranged from 0 to 7.5 mg/dl and the concentrations of PS were kept constant (10 mg/dl). Total RNA was harvested after 48 h of treatment. Fig. 3B shows that increased cholesterol content of liposomes could e¡ectively increase ICAM-1 mRNA expression. If the cholesterol:PS ratio was kept at 1:1, the increase in cholesterol correspondingly increased ICAM-1 mRNA (Fig. 3B).
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Fig. 4. Cholesterol transcriptionally activates ICAM-1 in HUVECs. (A) ICAM-1 mRNA decay in presence of actinomycin D. Con£uent HUVECs were treated with TNF-K (2 ng/ml) for 2 h followed by actinomycin D (5 mg/ml) with or without cholesterol, and total cellular RNA was isolated at 0, 1, 2, 4, and 6 h after treatment. ICAM-1 and GAPDH mRNA was detected by Northern blotting followed by densitometric measurement of the corresponding signal. The relative density of ICAM-1 was normalized with GAPDH. Results represent three separate experiments. (B) E¡ect of cholesterol on ICAM-1 promoter. Subcon£uent HUVECs were transfected with PGL3-0.98 plus pCMV-L-gal and then incubated for 24 h in EC medium with no supplementation (Ctrl) or with cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/ml). PMA (50 ng/ml) overnight treatment was used as positive control. Samples were collected and assayed for luciferase expression. The results were normalized against L-galactosidase. Data are presented as the mean þ S.D. of relative luciferase activities in ¢ve independent experiments, each performed in triplicate.
3.5. Cholesterol increases AP-1 binding activity
3.4. Cholesterol transcriptionally upregulates ICAM-1 To further explore the mechanism by which cholesterol upregulates ICAM-1 mRNA, we examined the upstream regulatory region of the ICAM-1 gene by transfecting HUVECs with an ICAM-1 promoterdriven reporter construct. Fig. 4B shows that cholesterol increases the promoter activity of the PGL30.98 construct by 2.65 þ 0.18-fold (P 6 0.05). To evaluate the ICAM-1 mRNA half-life in HUVECs after cholesterol treatment, we measured the time course of ICAM-1 decay in HUVECs after arrest of new transcription with actinomycin D (5 Wg/ml). HUVECs were pretreated with TNF-K (2 ng/ml) for 2 h to induce ICAM-1 expression. Total RNA was extracted after 0, 1, 2, 4, and 6 h of actinomycin D treatment and hybridized with probes for ICAM-1 or GAPDH. The half-life of ICAM-1 mRNA was 7 h with and without cholesterol exposure (Fig. 4B), which suggests that ICAM-1 mRNA stability was not a¡ected by cholesterol.
Con£uent HUVECs were exposed to cholesterolenriched liposomes for 2 h, 6 h and 12 h. The EMSA result in Fig. 5A shows that cholesterol increases AP1 binding as early as 2 h after exposure. The increased AP-1 binding activity remained high at all the time points examined. In contrast, NF-UB binding, which was markedly activated by PMA, was virtually absent in cholesterol-exposed HUVECs (Fig. 5B). As shown in Fig. 5C, only cholesterol-enriched liposomes increased AP-1 binding. Decreasing the cholesterol:PS ratio to 0.25:1 diminished this effect. In addition, cholesterol increased AP-1 binding in a dose-dependent fashion beginning at a cholesterol concentration of 2.5 mg/dl (Fig. 5D). 3.6. Cholesterol functionally increases AP-1-dependent protein expression Cholesterol increased AP-1 but not NF-UB binding as demonstrated by gel shift assays. To further
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Fig. 5. Cholesterol e¡ects on AP-1 and NF-UB binding by EMSA. HUVECs were exposed to cholesterol-enriched liposomes, and nuclear proteins were extracted. (A) and (B) Cholesterol:PS molar ratio 1:1, cholesterol concentration 5 mg/dl. (C) Cholesterol:PS with varying ratios. (D) Cholesterol with varying concentration for 6 h. EMSA was performed with use of an oligonucleotide of consensus AP-1 or NF-UB binding sequences as probe. Results are representative of three to four separate experiments.
determine whether cholesterol could speci¢cally activate an AP-1-driven promoter, we examined the effect of cholesterol by transfecting HUVECs with the pAP1-Luc construct or pNFUB-Luc construct. As shown in Fig. 6, cholesterol increased the promoter activities driven only by the AP-1 motif (P 6 0.05) but not those driven by the NF-UB motif. PMA, on the other hand, signi¢cantly activated both AP1- and NF-UB-dependent reporter luciferase activities (P 6 0.01). Thus, cholesterol speci¢cally upregulates genes driven by the AP-1 motif and has no e¡ect on the NF-UB-dependent reporter, which is highly responsive to PMA. 3.7. AP-1 is involved in cholesterol-induced upregulation of ICAM-1 The 5P-£anking region of the ICAM-1 gene con-
Fig. 6. E¡ect of cholesterol on expression of AP-1-driven or NF-UB-driven reporter gene. Subcon£uent HUVECs were cotransfected with pCMV-L-gal plus pAP1-Luc or pNFUB-Luc. After cholesterol-enriched liposomes or PMA (50 ng/ml) treatment, samples were collected and assayed for luciferase expression. The results were normalized against L-galactosidase. Data are presented as the mean þ S.D. of relative luciferase activities in ¢ve independent experiments.
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Fig. 7. E¡ect of cholesterol on ICAM-1 promoter region elements binding. HUVECs were pretreated with cholesterol-enriched liposomes (cholesterol:PS ratio 1:1, cholesterol concentration 5 mg/dl) for 2, 6, 12 h followed by nuclear extract isolation. PMA (50 ng/ ml) treatment was applied as a positive control. EMSA was performed with use of the oligonucleotide of ICAM-1 promoter region AP-1/Ets element (left panel) or NF-UB fragment (right panel) as probes. Results represent three separate experiments.
tains three putative AP-1-like sites and variant NFUB elements [21^24]. We used three synthesized oligo-DNA fragments containing the sequences of the three variant AP-1-like elements (AP1/TRE, 3321; AP-1/Ets, 3940; AP1, 31290) located in the 5P-untranslated region (UTR) of ICAM-1 gene as probes for EMSA [25]. Of the three AP-1-like elements, only the AP-1/Ets site responded to cholesterol exposure (Fig. 7, left panel). However, cholesterol did not increase binding activity of the ICAM-1 promoter region NF-UB site (3223), but did increase binding activity in the PMA-treated sample (Fig. 7, right panel). 4. Discussion This study is aimed at examining the hypothesis that cholesterol enrichment alone may induce EC activation, manifested by the regulation of adhesion molecules such as ICAM-1 expression in HUVECs. Cholesterol-enriched liposomes have been shown to transfer cholesterol to the cell membranes [26^28]. In this report, we utilized cholesterol-enriched liposomes as a cholesterol-loading system, to investigate the e¡ects of cholesterol enrichment on EC adhesion molecules, especially ICAM-1 regulation and upstream signaling. We demonstrated that (1) cholesterol-enriched liposome exposure increases the HUVEC cholesterol:phospholipid ratio, (2) cholesterol
transcriptionally upregulates ICAM-1, and (3) AP-1 and not NF-UB appears to play an important role in cholesterol-induced EC activation. This study shows, for the ¢rst time, that cholesterol, as a cellular function modulator, can activate ECs, likely through an AP-1-speci¢c pathway, leading to proin£ammatory and hence, potentially, pro-atherogenic changes. Cholesterol is an essential constituent of cells. Regulation of synthesis, in£ux and e¥ux keeps cellular cholesterol levels carefully controlled. Alterations in cellular cholesterol content have been shown to a¡ect membrane permeability, endocytosis, glucose transport, LDL binding, and cell cycle control [8,9, 29,30]. However, the role of cholesterol in activating ECs to a proin£ammatory state has not been studied. An objective in understanding the pathogenesis of hypercholesterolemia in atherogenesis is to determine how cholesterol enrichment of the endothelium affects EC function. The current study suggests that cholesterol can induce EC activation, likely through an AP-1 pathway, and can lead to ICAM-1 induction. Numerous articles, including our own, have demonstrated that LDL, a risk factor for atherosclerosis, is an EC activator [5,6,31]. LDL is a major carrier of cholesterol in vivo. Our previous study showed that LDL can activate ICAM-1 mainly through an AP-1mediated pathway [25]. One of the objectives in understanding LDL-induced atherogenesis is to determine the role of di¡erent moieties of the LDL
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particle and the mechanism by which they alter endothelial function. The present report extends our previous studies by focusing on cholesterol as a major stimulator. Striking similarities were observed in cholesterol- and LDL-induced EC activation. First, both can upregulate ICAM-1 expression. Second, both activate AP-1 but not NF-UB. Lastly, both enrich the EC cellular membrane with cholesterol. Our ¢nding indicates that many important aspects related to the role of LDL in atherosclerosis are directly linked to the accumulation of cholesterol in ECs. Adhesion molecules play a key role in the recruitment and extravasation of circulating leukocytes at the site of in£ammation [32]. ICAM-1 upregulation has been observed in lesional or lesion-prone areas of human atherosclerosis and hypercholesterolemic animals [33,34]. In ECs, ICAM-1 is upregulated in response to a wide variety of stimuli including proin£ammatory cytokines, LPS, PMA and H2 O2 . These stimuli increase ICAM-1 expression primarily through activation of ICAM-1 gene transcription [35]. Thus far, more than 4 kb of the 5P-£anking DNA of the human ICAM-1 gene has been cloned and sequenced [22,23]. Analysis of the 1.3-kb human ICAM-1 promoter region reveals multiple binding motifs for various transcription activators including three putative AP-1-like sites, variant NF-UB elements [21], AP-2, Ets, STATs and SP-1 sites, which suggests complex transcriptional regulation [22^24]. The variant NF-UB site in the promoter is responsive to cytokines and LPS and has been the target of several studies [36]. Sequence analysis indicates that AP-1 binding sites are recurrent elements in the promoters of many proin£ammatory genes such as ICAM-1, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). In the present study, we found that among the three AP-1-like elements in the ICAM-1 promoter region, the AP-1/Ets (3891 to 3908) site is most responsive to cholesterol exposure. However, we did not observe increased binding of the NF-UB-like element, which is consistent with our previous ¢nding in LDL-induced ICAM-1 transcriptional activation [6]. Both AP-1 and NF-UB transcription factors have been reported to be involved in the regulation of ICAM-1 [21,24,36^38]. In ECs, deregulation of AP1 can be seen in response to a variety of pathophysiologic stimuli such as cytokines [39], bacterial en-
dotoxin, antioxidants [40], hypoxia [41], and £uid shear stress [42]. Particularly, LDL induces a sustained activation of AP-1 in ECs [6,43,44]. Although NF-UB has been shown to play an important role in EC activation [45^47], increasing evidence indicates the importance of AP-1 in endothelial homeostasis and dysfunction. We recently demonstrated the induction of ICAM-1 and MCP-1 independent of NF-UB activation [25]. Overexpression of AP-1 components alone directly causes ICAM-1 induction in ECs [25], which con¢rms that AP-1 itself is su¤cient to cause phenotypic activation and induction of ICAM-1 in ECs. In contrast to most proin£ammatory cytokines, growth factors and bacterial endotoxin, cholesterol has no e¡ect on NF-UB. Interestingly, this result is consistent with our previous ¢nding that LDL activates AP-1 but not NF-UB in ECs [6]. Further, AP-1- and NF-UB-driven promoter transfections revealed that AP-1 activation is mainly responsible for cholesterol-induced EC activation, and NFUB appears not to be involved in this process. Cholesterol appears to activate ICAM-1 predominantly through an AP-1-mediated mechanism. Similarly, a stimulus-speci¢c NF-UB-independent mechanism was reported in the induction of the IL-8 gene [48]. Cholesterol appears to be an endothelial agonist distinct from other cell stimulators such as cytokines, endotoxin, oxidized LDL, and phorbol esters by activating ECs predominantly through transcription factor AP-1. Thus, enrichment of cholesterol, as a chronic vascular agonist, may perturb the EC phenotype through a mechanism that is distinct from an acutely activated pathway. A correlate to this is that atherosclerotic plaque formation is a chronic process, in keeping with the present results. The mechanistic basis for the e¡ect of cholesterol enrichment on cellular function modulation can be interpreted from the actions of cholesterol on the membrane lipid bilayer. Cholesterol is non-randomly distributed between lea£ets of the membrane bilayer, and the cholesterol distribution in membranes is highly dynamic. This o¡ers numerous possibilities for explaining the in£uence of cholesterol on membrane-embedded proteins and subsequent signal transduction. Cholesterol e¡ects can be caused by its modi¢cation of cellular membrane £uidity, which has been seen in red blood cells, hepatocytes, platelet, epithelial cells and SMCs [29]. Prolonged incuba-
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tion of ECs with native LDL is associated with marked reduction in relative EC plasma membrane £uidity [5]. Likewise, exposure of ECs to cholesterol may decrease cell membrane £uidity, perturb the cell surface receptors, and thereby a¡ect intracellular signaling. In conclusion, we demonstrate, for the ¢rst time, that cholesterol enrichment of ECs activates the AP1 signaling pathway, resulting in an increase in ICAM-1 expression. This leads to the induction of endothelial adhesion molecule upregulation and thus EC activation. This ¢nding is consistent with the hypothesis that cholesterol enrichment of ECs mediates initiation of atherogenesis following the development of in vivo serum hypercholesterolemia. To our knowledge, this is the ¢rst evidence of the crucial role of the cholesterol molecule in EC activation. This ¢nding may contribute to our understanding of the mechanisms of hypercholesterol-induced EC dysfunction.
[7] [8] [9] [10]
[11]
[12] [13]
[14]
[15]
Acknowledgements This study was supported by the NIH grant HL43023 (to M.B.S.). We thank Dr. D. Johnson for his thoughtful critiques of the manuscript. We thank Dr. J.A. Thompson for providing recombinant human ¢broblast growth factor.
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