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From the Laboratory for Systems Biology and Medicine (W.T., T.K., N.N.), Research Center for Advanced Science and Technology, The University of Tokyo, Japan; Chugai Pharmaceutical Co Ltd (W.T.), Shizuoka, Japan; the Department of Pharmacology and Toxicology (Y.K., A.C.), Kyorin University School of Medicine, Tokyo, Japan; and the Departments of Pathology (N.S., M.K.) and Neurology (S.T.), Tokyo Women’s Medical University, Tokyo, Japan.
ABSTRACT
Objective— A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. We performed DNA microarray experiments to explore novel responses of human umbilical vein endothelial cells (HUVECs) to oxLDL and its components.
Methods and Results— cDNA microarray analysis showed that oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2-nonenal, and oxysterols altered gene expression specifically, but some genes were commonly induced in HUVECs. Solute carrier family 3 member 2 and family 7 member 5, encoding the heavy chain of the cell surface antigen 4F2 (4F2hc) and the L-type amino acid transporter 1 (LAT1), respectively, were induced by oxLDL and many oxidation products. LAT1 requires 4F2hc to form a heterodimeric functional complex to transport neutral amino acids into the cell. LysoPC increased membrane protein levels of LAT1 confirmed by Western blot analysis and also uptake of L-leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor.
Conclusions— These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells.
Key Words: amino acid transporter ? atherosclerosis ? cytokine ? HUVEC ? LysoPC
Introduction
Atherosclerosis, which leads to coronary heart disease and stroke, is the most common cause of death in industrialized nations. It has been suggested that oxidative modification of low-density lipoprotein (LDL) is a key initial event in atherosclerosis pathogenesis,1 and a wide variety of oxidized lipids have been detected in atherosclerotic lesions.2 LDL is composed of a cholesteryl ester (CE) and triglyceride core with an outer monolayer composed of phosphatidylcholine (PC) and free cholesterol solubilized in blood by 1 molecule of apolipoprotein.3 The esterified fatty acids of PC and CE are oxidized enzymatically and nonenzymatically to yield lipid hydroperoxides as the primary products,4 followed by secondary reactions to form lipid hydroxides and aldehydes such as malondialdehyde, acrolein,5 and 4-hydroxy-2-nonenal (4HNE). Acrolein and 4HNE are known to be highly reactive and to form adducts with proteins and nucleic acids.6 In particular, many studies have shown that 4HNE regulates cell-signaling pathways through activation protein 1 (AP-1).7–9 Cholesterol is also oxidized to give several classes of oxysterols: 7-ketocholesterol, which induces monocyte differentiation and promotes foam cell formation;10 22(R)-hydroxycholesterol, which is a ligand for the liver X receptor and regulates the expression of genes involved in cholesterol and fatty acid homeostasis;11 and 25-hydroxycholesterol, which regulates cholesterol synthesis via the sterol regulatory element-binding protein (SREBP)/SREBP cleavage-activating protein regulatory pathway.12 Lysophosphatidylcholine (LysoPC) is present at high concentrations in oxidized LDL (oxLDL) and formed via the reaction of phospholipase A2. -Palmitoyl-LysoPC (16:0) is known to induce various protein kinases in vascular cells, including protein kinase C (PKC), extracellular signal regulated kinase (ERK) 1 and 2 (ERK1/2), and p38.13–16
We performed large-scale gene expression analysis using human endothelial cells exposed to oxLDL and lipid oxidation products such as LysoPC, 4HNE, 7-ketocholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol contained in oxLDL. There were several genes that were commonly induced by oxLDL and some of the oxidation products, but they were assumed to be important from the therapeutic point of view. In this article, we report the induction of solute carrier genes that encode for an amino acid transporter subfamily.
Amino acid transporters in the plasma membrane mediate the uptake of nutrients into cells. Among the documented amino acid transport systems, system L is a major transport system for the Na+-independent nutrient uptakes, whereas 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is involved in the sensitive transport of large neutral amino acids.17
The L-type amino acid transporter 1 (LAT1) belongs to system L and requires the heavy chain of the cell surface antigen 4F2 (4F2hc) for functional expression. It transports neutral amino acids, most of which are essential amino acids.18–20 Although the expression of 4F2hc is apparently ubiquitous, LAT1 is expressed in the brain, placenta, testes, bone marrow, fetal liver, and peripheral leukocytes.18,21 Furthermore, LAT1 is highly expressed in certain tumor cell lines18 as well as in malignant tumors.22 Because overexpression of LAT1 supports the high protein synthesis rates in growing cells, this finding has given rise to the suggestion that LAT1 might be a useful therapeutic target. The consequence of the increased protein synthesis in endothelial cells should be another important issue to be investigated. This article reports for this first time that increased LAT1 expression can be induced by lipid oxidation products relevant to inflammatory responses in atherogenesis.
Methods
Cell Culture and Treatment
Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were obtained from a commercial source (Clonetics, San Diego), and all experiments were conducted in 4 passages. HUVECs and HAECs were grown in endothelial cell growth factor-containing medium-2 (EGM-2; Clonetics) with 2% FBS (Clonetics) at 37°C in a 5% CO2 atmosphere. After reaching confluence, the medium was changed to EGM-2 with 2% FBS containing 200 μg/L oxLDL, 10 μmol/L 7-ketocholesterol (Steraloids Inc), 10 μmol/L 22(R)-hydroxycholesterol (Sigma), 10 μmol/L 25-hydroxycholesterol (Sigma), 30 μmol/L -palmitoyl-LysoPC (Sigma), or 5 μmol/L 4HNE (Cayman Chemical). All chemicals were dissolved in ethanol (EtOH; Wako), which was diluted with EGM-2, resulting in a final EtOH concentration of 0.01%. The control cells were cultured in EGM-2 containing 0.01% EtOH in the absence of oxidation products.
Details for methods of measurement of lipid oxidation products in oxLDL, Northern blot and real-time polymerase chain reaction (PCR) analysis, Western blot analysis, L-leucine uptake, measurement of cytokines, immunohistochemical study, adhesion molecule measurement, and others are in the expanded Methods section available online at http://atvb.ahajournal.org.
Results
Effect of oxLDL and Its Components on Gene Expression in HUVECs
In preliminary experiments, concentrations of lipid oxidation products were first assessed from samples of oxLDL (200 μg/mL) oxidized for 18 hours with 100 μmol/L copper. This treatment results in extensively oxidized LDL and allows for a feasible assessment of the maximum concentrations of individual components likely to stimulate a biological response. No toxicity was evident under any conditions for 24 hours as assessed by crystal violet staining and trypan blue exclusion method (data not shown).
Gene expression was determined in HUVECs using the GeneChip human genome focus array, which contains 8794 genes. Expression was determined in response to 200 μg/mL oxLDL, 10 μmol/L 7-ketocholesterol, 10 μmol/L 22(R)-hydroxycholesterol, 10 μmol/L 25-hydroxycholesterol, 30 μmol/L LysoPC, and 5 μmol/L 4HNE. The concentration of each oxidation product was determined according to that of oxLDL used in the present study.
Whereas oxLDL, LysoPC, and 4HNE induced expression of 117 genes, 105 genes, and 14 genes, respectively, a few genes were responsive to 3 different kinds of oxysterols: 0 7-ketocholesterol, 5 22(R)-hydroxycholesterol, and 1 25-hydroxycholesterol, respectively. The table shows the genes that were upregulated by 4 hours of treatment with oxLDL >2-fold. The fold change values obtained by treatment with oxidation products are also shown. Among these upregulated genes, the solute carrier family (SCL) genes were induced not only by oxLDL but also LysoPC, 4HNE, and 22(R)-hydroxycholesterol. SCL genes are known to encode transporter subunits. Another interesting gene induced by oxLDL, LysoPC, 4HNE, and 22(R)-hydroxycholesterol was the CCAAT/enhancer-binding protein ? (C/EBP?), a nuclear factor for interleukin 6 (IL-6) and IL-8 expression.
Upregulated Genes by oxLDL and its Components
Induction of mRNA of SLC3A2 and SLC7A5 by oxLDL
In the next series of experiments, we verified the accuracy of the GeneChip findings by Northern blot analysis for SLC3A2 and SLC7A5. HUVECs were exposed to extensively oxidized LDL (20 or 200 μg/mL) for up to 24 hours. Figure 1A shows that oxLDL induced the expression of both SLC3A2 and SLC7A5 in a time- and concentration-dependent manner for up to 8 hours. The concentration of LysoPC in this oxLDL was measured and found to be 66.8 (mol/mol LDL). Thus, the concentration of LysoPC in medium was 30 μmol/L when 200 μg/mL oxLDL was added into medium. We measured other oxidation products in this oxLDL and calculated the concentration of each of them in medium as follows: 4.4 μmol/L 7-ketocholesterol, 2.7 μmol/L 22(R)-hydroxycholesterol, 5.4 μmol/L 25-hydroxycholesterol, and 0.5 μmol/L 4HNE.
Figure 1. Time- and concentration-dependent induction of mRNA expression of SLC3A2 and SLC7A5 by oxLDL and its components. A, Northern blot analysis was used to determine treatment effect of HUVECs with 20 or 200 mg/mL oxLDL for up to 24 hours on mRNA expression levels of SLC3A2 and SLC7A5. B, Induction of SLC3A2 and SLC7A5 by oxidized LDL components in HUVECs was examined. HUVECs were treated by 10 μmol/L 7-ketocholesterol (7keto), 10 μmol/L 22(R)-hydroxycholesterol (22(R)OH), 10 μmol/L 25-hydroxycholesterol (25OH), 30 μmol/L LysoPC, or 5 μmol/L 4HNE for 4 hours. All chemicals were dissolved in EtOH. 28.5 and 18.5 indicate ribosomal RNA. Real-time PCR analysis were performed for SLC3A2 (C) and SLC7A5 (D) expression induced by LysoPC in HUVECs and HAECs. All data obtained were normalized by GAPDH values and shown as the mean±SD (n=3) of the ratio against time 0. *P<0.05; **P<0.01; ***P<0.005.
Induction of mRNA of SLC3A2 and SLC7A5 by oxLDL Components
HUVECs were treated with 10 μmol/L 7-ketocholesterol, 10 μmol/L 22(R)-hydroxycholesterol, 10 μmol/L 25-hydroxycholesterol, 30 μmol/L LysoPC, and 5 μmol/L 4HNE. The microarray data showed that the fold change of SLC3A2 induced by LysoPC, 4HNE, or 22(R)-hydroxycholesterol was 1.97±0.29, 2.79±0.48, and 2.42±0.36, respectively, and that of SLC7A5 by LysoPC, 4HNE, or 22(R)-hydroxycholesterol was 4.58±2.10, 1.86±0.92, and 1.95±0.47, respectively. These results agree with the results of the Northern blot analysis (Figure 1B).
Because LysoPC is one of the most abundant oxidation products in oxLDL and induced SLC genes most extensively, the time-dependent induction of mRNA of SLC3A2 and SLC7A5 in HUVECs treated with 30 μmol/L LysoPC was followed for up to 4 hours by quantitative real-time PCR (Figure 1C and 1D). All data normalized by GAPDH and cyclophilin showed almost the same results. The expression of SLC3A2 and SLC7A5 was increased over time. The same experiments were performed for HAECs and showed that LysoPC induced mRNA both of SLC3A2 and SLC7A5 in aortic endothelial cells in a time-dependent manner.
Induction of Amino Acid Transporter Protein by LysoPC
We confirmed an increase in LAT1 (SLC7A5) protein level in membrane fraction of HUVECs after exposure to 30 μmol/L LysoPC for 6 hours (Figure 2). The 125-kDa protein corresponding to a heterodimeric complex of LAT1 and 4F2hc was detected under the nonreducing condition. The band of 38-kDa protein corresponding to LAT1 monomer appeared by reducing the protein complex with 2-mercaptoethanol.
Figure 2. Increase in protein levels of LAT1 induced by LysoPC. Western blot analysis using a monoclonal antibody against LAT1 was performed for the membrane fraction (5 μg of protein) prepared from HUVECs after treatment with or without 30 μmol/L LysoPC for 6 hours. The 125-kDa protein band detected in the nonreducing condition shifted to a 38-kDa protein band by the treatment of 2-mercaptoethanol (2ME).
Effect of LysoPC on L-Leucine Uptake in HUVECs
The effect of LysoPC on amino acid transport in HUVECs was examined using L-leucine (Figure 3). LysoPC increased L-leucine uptake significantly after 6 hours of incubation, the effect of which was almost completely inhibited by 1 mmol/L BCH, a selective inhibitor of system L amino acid transporter (LAT1, LAT2, and LAT3) under Na+-free conditions. The GeneChip data showed that SLC7A8 encoding LAT2 was not expressed in HUVECs and also was not induced by LysoPC (1.30±0.30). SLC43A1 (named prostate cancer overexpressed gene 1) encoding LAT3 was expressed slightly in HUVECs but was not induced by LysoPC (0.85±0.13). To confirm it, the following experiment was performed. Because LAT3 has been shown not to transport L-histidine or L-tryptophan,23 a competitive experiment was performed using 1 mmol/L L-leucine, L-histidine, and L-tryptophan. The uptake of L-leucine induced by LysoPC was competitively inhibited by these amino acids. These results suggest that the increase in L-leucine uptake induced by LysoPC was attributable to pronounced LAT1 activation.
Figure 3. The effect of LysoPC on L-leucine uptake in HUVECs. Induction of L-leucine transport in HUVECs treated with 30 μmol/L LysoPC for 6 hours (A). HUVECs were incubated in the Na+-free uptake solution containing 10 μmol/L L-leucine for 1 minute, and the L-leucine uptake was measured in the presence or absence of 1 mmol/L BCH, 1 mmol/L L-leucine (L-Leu), 1 mmol/L L-histidine (L-His), and 1 mmol/L L-tryptophan (L-Trp) (B). Each was performed in triplicate. *P<0.05 vs no addition and **P<0.005 vs LysoPC.
Contribution of SLC3A2 and SLC7A5 Expression to Cytokine Production in HUVECs
It has been reported that LAT1 is upregulated in malignant tumors, and its expression is related to the growth and proliferation of tumor cells. Therefore, we investigated whether cell proliferation would be enhanced after exposure of HUVECs to LysoPC. An enhancement of cell proliferation by LysoPC was not observable, at least as assessed by crystal violet assay and trypan blue assay (data not shown).
To find consequences of amino acid uptake into cells, cytokines released into the culture medium were measured using a BioPlex cytokine analyzer, which has the capacity to measure 17 distinct cytokines. HUVEC exposure to 30 μmol/L LysoPC for 24 hours significantly increased the release of IL-6 and IL-8 into medium (Figure 4). Release of IL-6 and IL-8 from HUVECs after exposure to LysoPC was inhibited by BCH by 40% and 50%, respectively. BCH did not affect basal levels of IL-6 and IL-8 in the absence of LysoPC, suggesting that a substantial part of the increase in production of IL-6 and IL-8 induced by LysoPC was attributable to LAT1. IL-6 and IL-8 might also be produced using endogenous intracellular pools of amino acids in HUVECs on stimulation by LysoPC, and thus would not be inhibited by the transporter inhibitor. No release of the cytokines IL-1?, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p70, IL-13, IL-17, macrophage inflammatory protein 1-?, interferon (INF-), granulocyte-colony stimulating factor (CSF), granulocyte-macrophage CSF, tumor necrosis factor , or monocyte chemoattractant protein 1 was detected after exposure of HUVECs to LysoPC (data not shown).
Figure 4. Cytokine release induced by LysoPC. HUVECs were incubated with 30 μmol/L LysoPC for 24 hours. The culture medium was collected, and cytokines were measured by using Bio-Plex Cytokine Assay Kit in the absence or presence of 1 mmol/L BCH. Results for IL-6 (A) and IL-8 (B) (mean±SD; n=6) are shown. *P<0.005 vs no addition; **P<0.005 vs LysoPC; ***P<0.01 vs LysoPC.
Detection of LAT1 in LDL Receptor Knockout Mouse Aorta
To test induction of LAT1 expression in atherogenic animals, immunohistochemical study using a monoclonal antibody against LAT1 was performed for aortas of LDL receptor knockout mice fed with high-fat or normal diet (Figure 5). LAT1 was detected predominantly in endothelial cells and macrophages but not smooth muscle cells in ascending aortas of mice on a high-fat diet compared with mice on a normal diet.
Figure 5. Detection of LAT1 in aorta of LDL receptor knockout mouse. The aortas of LDL receptor knockout mice that were fed a normal diet (A) and a high-fat diet (B) were stained by a monoclonal antibody raised against LAT1. LAT1 was expressed in endothelial cells of mice on a high-fat diet but not with a normal diet. LAT1 was also expressed in macrophages accumulating in the intima of aortas of mice fed a high-fat diet (C).
Discussion
OxLDL contains a variety of lipid oxidation products derived from phosphatidylcholine, CEs, fatty acids, and cholesterol. LysoPC is thought to be one of the major oxidation products of oxLDL, and its biological function in vascular cells has been investigated extensively.13–16 A number of studies have shown that LysoPC stimulates endothelial cells to promote expression of adhesion molecules24–26 and release cytokines.27,28 A large-scale analysis of gene expression in HUVECs after exposure to oxLDL and lipid oxidation products including LysoPC revealed the unexpected finding that oxLDL and its components such as LysoPC affect the functions of endothelial cells by enhancing amino acid transport into cells.
SLC3A2 and SLC7A5 are translated into the heavy chain of 4F2hc and LAT1, respectively. These proteins form a functional complex for the transport of large neutral amino acids into the cell. Because LAT1 has been shown to be expressed highly in certain cancer cell lines18 as well as malignant tumors,22 its contribution to cell proliferation is strongly implicated. The enhancement of proliferation is more important in the smooth muscle cells than endothelial cells in the vascular wall but may also play a role in the balance between endothelial cell growth and apoptosis in response to injury. Because LAT1 activity regulation has become a target in cancer therapy, a specific inhibitor for LAT1 is now under active investigation. This article reports evidence for a novel function of oxLDL and its components in atherogenesis and suggests that LAT1 may prove to be a useful target molecule in inflammatory diseases.
Among the 17 cytokines measured, IL-6 and IL-8 were induced by LysoPC treatment in HUVECs. There is a binding site for C/EBP? in the promoter region of IL-6 and IL-8.29–33 The induction of C/EBP? mRNA by LysoPC was examined by a GeneChip experiment (Table, fold change 6.59±2.54), and the evident genetic activity may account for a substantial part of this increased production of IL-6 and IL-8. C/EBP? mRNA was also increased by oxLDL, 4HNE, and 22(R)-hydroxycholesterol. In addition, GeneChip analysis revealed that the mRNA level of IL-6 was significantly increased by LysoPC (fold change 2.35±0.35). However, a significant induction of IL-8 mRNA could not be confirmed because its basal level was too low to evaluate and allow statistical analysis. According to computer analysis of the ideal transcription factor binding sites in the promoter region, 4 C/EBP? binding sites were found within 2000 bp in the promoter region of both SLC3A2 and SLC7A5. The molecular mechanisms by which amino acid transporter gene expression is enhanced by LysoPC have yet to be elucidated, but overexpression of the transporter may account in part for the proinflammatory effects of LysoPC. It has been reported that the promoter region of SLC3A2 displays sequence homologies with IL-2 and the IL-2 receptor chain, the induction of which is important for T-cell activation.34,35 In contrast to cytokines, a relationship between amino acid transport and adhesion molecule expression was not observed (see online supplement).
Computer analysis suggests that in the promoter region, there are certain other transcription factor-binding sites such as AP-1, cAMP response element-binding protein (CREB), SREBP, and specificity protein 1 (Sp1) for SLC3A2; and in addition to them, there is a nuclear factor B (NF-B) site in that of SLC7A5. Several signaling pathways active in endothelial cells have been identified after exposure to LysoPC. Phosphorylation of CREB by LysoPC is reported in bovine arterial endothelial cells,36,37 and NF-B activation has been shown to occur in response to LysoPC and prevented by protein tyrosine kinase inhibitors but not by cAMP-dependent protein kinases or PKC inhibitors.26 However, Sugiyama et al report that NF-B activation by LysoPC is concentration-dependent, biphasically regulated, and PKC activation might be involved in part in the LysoPC-induced NF-B activation in HUVECs.13 They also have shown that LysoPC increases the activities of AP-1 and CREB but not Sp1 and that only AP-1 activation in their experiments was PKC dependent. In addition to the PKC pathway, the mitogen-activated protein (MAP) kinases (ERK1/2) and the c-Jun N-terminal kinases are known to act as AP-1 activators.13,38,39 The overexpression of dual-specificity phosphatase 1 suggests activation of MAP-kinase pathways (Table).40 The signaling pathway crucial for the LysoPC-induced expression of SLC3A2 and SLC7A5 is under continuing investigation.
Acknowledgments
This work was supported in part by a grant-in-aid of Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology. We thank Drs. Yasukazu Yoshida, Satoshi Yamada, and Koichi Sumi for helping measurements of LysoPC, HNE-adduct, and cytokines, respectively. We also thank Akashi Izumi, Akiko Kikuchi, and Chizuru Nagao for excellent technical assistance.
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