HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, Y. W. Articles by Toborek, M. Search for Related Content PubMed PubMed Citation Articles by Lee, Y. W. Articles by Toborek, M. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 42, 783-791, May 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Interleukin 4 induces transcription of the 15-lipoxygenase I gene in human endothelial cells

Correspondence to: Michal Toborek, To whom correspondence should be addressed., mjtobo00{at}pop.uky.edu (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The reticulocyte-type 15-lipoxygenase (15-LO-I) has been implicated in atherogenesis because of its capability of oxidizing low density lipoprotein. Therefore, we investigated the expression of the 15-LO-I gene in human umbilical vein endothelial cells (HUVEC). Nonactivated HUVEC did not exhibit detectable 15-LO-I mRNA. However, exposure of the cells to interleukin 4 (IL-4) induced the transcription of the 15-LO-I gene in a time- and concentration-dependent manner. Interestingly, this induction was not paralleled by a concomitant production of the functional 15-LO-I enzyme, as indicated by activity assays and immunoblotting. To gain more information about the mechanism of the induction process, we investigated IL-4-dependent activation of nuclear transcription factors for which binding sites were previously identified in the 5'-flanking region of the human 15-LO-I gene. Electrophoretic mobility shift assays revealed that IL-4 can activate signal transducer and activator of transcription 6, activator protein 2, GATA motif-binding transcription factor 1, nuclear factor 1, and SP-1 in HUVEC in a time- and concentration-dependent manner. Activation of these transcription factors was observed as early as 30 min after cytokine exposure.

These data indicate that IL-4 upregulates the transcription of the 15-LO-I gene in human vascular endothelial cells, and this process may involve the activation of several nuclear transcription factors. The lack of active 15-LO-I protein in the presence of functional 15-LO-I mRNA suggests additional regulatory elements of 15-LO expression at posttranscriptional levels. — Lee, Y. W., H. Kühn, S. Kaiser, B. Hennig, A. Daugherty, and M. Toborek. Interleukin 4 induces transcription of the 15-lipoxygenase I gene in human endothelial cells. J. Lipid Res. 2001. 42: 783;–791.

Supplementary key words: cytokines, atherosclerosis, oxidation, transcriptional regulation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The reticulocyte-type 15-lipoxygenase (15-LO-I) is a lipid-peroxidizing enzyme that converts free and/or esterified polyunsaturated fatty acids (e.g., linoleic acid or arachidonic acid) to hydroperoxy derivatives, such as (13S)-hydroperoxy-9Z,11E-octadecadienoic acid and 15-hydroperoxy-5Z,8Z, 11Z,13E-eicosatetraenoic acid (15-HPETE). In cellular systems 13-hydroperoxy-9Z,11E-octadecadienoic acid and 15-HPETE are rapidly reduced to 13-hydroxy-9Z, 11E-octadecadienoic acid and 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE), respectively (1). 15-LO-I has been implicated in atherogenesis (2) (3). This hypothesis was based on the observations that the purified enzyme is capable of oxidizing low density lipoproteins (LDL) to an atherogenic form (4) (5) and that 15-LO-I protein colocalizes in atherosclerotic lesions with epitopes of oxidized LDL (6). The findings that a specific 15-LO-I inhibitor attenuated the development of atherosclerosis in cholesterol-fed rabbits (7) and that 12/15-LO-I knock-out mice develop less pronounced atherosclerosis when crossed with apolipoprotein E knock-out mice (8) appear to support this proatherogenic hypothesis.

Because of the pathophysiological importance of 15-LO-I in atherosclerosis, the regulation of LO expression has developed into a major field in lipoxygenase research. Several lines of experimental evidence indicate that the expression of 15-LO-I is regulated at transcriptional, translational, and posttranslational levels (9). In immature red blood cells, the 15-LO-I mRNA is present as translational inactive ribonucleotide-protein particles, which become translationally active at later stages of cell maturation (10). The activation process involves the proteolytic breakdown of specific mRNA-binding proteins, which were shown to be responsible for translational inhibition (11) (12). In contrast to the translational regulation of 15-LO-I mRNA, little is known about the regulatory mechanisms at transcriptional levels. It was reported that the pleiotropic cytokine interleukin 4 (IL-4) induces the expression of the 15-LO-I in human peripheral monocytes (13), in alveolar macrophages (14), in colorectal carcinoma cells (15), in WI-26 pulmonary epithelial cells (16), and in the lung carcinoma cell line A549 (17). The functionally related cytokine IL-13 also induced 15-LO-I expression in human peripheral monocytes (18), in A549 cells (17), and in murine peritoneal macrophages (19). Although detailed mechanisms of cytokine-mediated 15-LO-I induction are currently unknown, transcriptional regulation may be of particular importance. Structural analysis of the 5'-flanking region of the 15-LO-I gene revealed the existence of potential binding sites for a variety of transcription factors, such as signal transducer and activator of transcription 6 (STAT6), activator protein 2 (AP-2), GATA motif-binding transcription factor 1 (GATA-1), nuclear factor 1 (NF-1), and SP-1 (20) (21).

IL-4 is an immunomodulatory cytokine, which is secreted by helper T cell type 2 (Th2) lymphocytes, eosinophils, and mast cells (22) (23). It promotes differentiation of premature lymphocytes to Th2 cells and induces immunoglobulin class switching in B lymphocytes. It is present in elevated concentrations in tissues of patients with chronic inflammatory disorders (21) (24). IL-4 may also be involved in atherogenesis because increased levels of this cytokine were found in atherosclerotic vessels (25) and IL-4 is capable of inducing endothelial expression of vascular cell adhesion molecule 1 (26).

Although IL-4-dependent 15-LO-I expression has been studied in various cellular systems (12) (13) (14) (15) (16) (17) (18), it remained unclear whether 15-LO-I can be induced by this cytokine in endothelial cells. In the present study we investigated the effects of IL-4 on the expression of the 15-LO-I gene in human umbilical vein endothelial cells (HUVEC) and found that IL-4 induces the transcription of this gene. Moreover, we observed an activation of several transcription factors, for which putative binding sites exist in the 5'-flanking region of the human 15-LO-I gene.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation and culture of HUVEC
HUVEC were isolated and cultured as described previously (27). Cells were determined to be endothelial by their cobblestone morphology and uptake of fluorescently labeled acetylated LDL (1,1'-dioctadecyl-3,3,3'3'-tetramethyl-indocarbocyanine perchlorate; Molecular Probes, Eugene, OR). HUVEC from passage 2 were used in the present study. Cultures were treated with IL-4 (up to 50 ng/ml) in experimental media containing 10% fetal bovine serum.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from HUVEC by the use of TRI reagent (Sigma, St. Louis, MO) according to the manufacturer protocol. A standard RT reaction was performed at 42°C for 60 min in 20 µl of 5 mM MgCl₂, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP, recombinant RNasin ribonuclease inhibitor (1 U/µl), avian myeloblastosis virus reverse transcriptase (15 U/µg), and 0.5 µg of oligo(dT)₁₅ primer (Promega, Madison, WI). For quantitation, expression of mRNA of the human 15-LO-I gene was related to mRNA of ß-actin (a housekeeping gene). For amplification of the 15-LO-I gene and the ß-actin gene, the following primer combinations were used: 5'-GAG TTG ACT TTG AGG TTT CGC-3' and 5'-GCC ACG TCT GTC TTA TAG TGG-3' (15-LO-I; expecting 952-bp fragment) and 5'-AGC ACA ATG AAG ATC AAG AT-3' and 5'-TGT AAC GCA ACT AAG TCA TA-3' (ß-actin; expecting 188-bp fragment). Two microliters of the reverse transcriptase reaction was used for PCR amplification. The PCR mixture consisted of a Taq PCR master mix kit (Qiagen, Valencia, CA) and 20 pmol of primer pairs in a total volume of 50 µl. Thermocycling was performed according to the following profile: 94°C for 45 s, 60°C for 45 s, and 72°C for 2 min, repeated 35 times and followed by a final extension at 72°C for 7 min. Amplification was linear within the range of 25;–40 cycles. PCR products were separated by 2% agarose gel electrophoresis, stained with SYBR^® Green I (Molecular Probes), and visualized by phosphoimaging technology (FLA-2000; Fuji, Stamford, CT).

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts from HUVEC were prepared according to the method of Beg et al. (28). The DNA-binding consensus or mutant sequences for various transcription factors were purchased as double-stranded oligonucleotides from Santa Cruz Biotechnology (Santa Cruz, CA). The following oligonucleotides were used [all sequences are from 5' to 3'; only the sense strand is shown; the consensus sequences are underlined and mutated (mt) nucleotides are differentiated by lower case]: STAT6, GTA TTT CCC AGA AAA GGA AC; mt STAT6, GTA TTT Cgg ttA AAA GGA AC; AP-2, GAT CGA ACT GAC CGC CCG CGG CCC GT; mt AP-2, GAT CGA ACT GAC CGC ttG CGG CCC GT; GATA-1, CAC TTG ATA ACA GAA AGT GAT AAC TCT; mt GATA-1, CAC TTc tTA ACA GAA AGT ctT AAC TCT; NF-1, TTT TGG ATT GAA GCC AAT ATG ATA A; mt NF-1, TTT TGG ATT GAA taa AAT ATG ATA A; SP-1, ATT CGA TCG GGG CGG GGC GAG C; mt SP-1, ATT CGA TCG Gtt CGG GGC GAG C. Oligonucleotides were end labeled with [-³²P]ATP and T4 polynucleotide kinase. Competition studies were performed by the addition of a molar excess of unlabeled oligonucleotide to the binding reaction. Supershift experiments were performed by the addition of antibody to the binding reaction for 25 min before the addition of labeled oligonucleotide probe. Rabbit polyclonal antibodies to STAT6 and NF-1 and goat polyclonal antibodies to AP-2, GATA-1, and SP-1 were purchased from Santa Cruz Biotechnology. Resultant protein-DNA complexes were electrophoresed on native 5% polyacrylamide gels using 0.5x TBE buffer [0.1 M Tris-HCl, 90 mM boric acid, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.4] at 150 V for 2 h, dried under vacuum, and autoradiographed with intensifying screens.

Western blot
Western blot was performed as described previously (29). Briefly, treated endothelial cells were scraped from 60-mm-diameter dishes in 150 µl of sample buffer (2 mM EDTA, 2.3% sodium dodecyl sulfate, 10% glycerol, and 62 mM Tris, pH 6.8). Protein concentrations were determined as described by Lees and Paxman (30). Equal amounts (up to 20 µg) of total protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% acrylamide) and transferred to a nitrocellulose membrane at 250 mA for 1.5 h at 4°C. Nonspecific binding was blocked by overnight incubation of the nitrocellulose membrane with 5% fat-free milk in Tris-buffered solution at 4°C. Sheep anti-15-LO-I polyclonal antiserum (Cayman Chemicals, Ann Arbor, MI) was used as primary antibody. This antibody was generated against rabbit reticulocyte 15-LO-I, but it cross-reacted with human 15-LO-I. Anti-sheep IgG labeled with horseradish peroxidase was used as a secondary antibody. The chemiluminescence emitted from luminol oxidized by horseradish peroxidase was detected with an enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ) and Eastman Kodak (Rochester, NY) XAR-5 film for autoradiography.

15-LO-I activity and high performance liquid chromatography (HPLC) measurements
Treated endothelial cell cultures were harvested, suspended in 1 ml of phosphate-buffered saline, and sonicated. Arachidonic acid or linoleic acid was then added at the final concentration of 160 µM and lysates were incubated for 15 min at 37°C. Cellular lipids were extracted with chloroform;–methanol;–water 2:1:1 (v/v/v). After the reduction of lipid peroxides, aliquots (0.5 ml) of cell extracts were analyzed by reversed-phase HPLC on a Nucleosil C₁₈ column (KS-system, 250 x 4 mm, 5 µm; Macherey-Nagel, Dueren, Germany). A solvent system of methanol;–water;–acetic acid 80:20/0.1 (v/v/v) was used at a flow rate of 1 ml/min, and the absorbance at 235 nm was recorded. The chromatographic scale was calibrated by injecting known amounts of 15-HETE.

To obtain more detailed structural information about the HETE positional isomers, products comigrating with the different HETE isomers in reversed-phase HPLC were analyzed by straight-phase HPLC. Straight-phase HPLC was performed on a Zorbax-SIL column (250 x 4 mm, 5 µm) with a solvent system of n-hexane;–2-propanol;–acetic acid 100:2:0.1 (v/v/v; flow rate, 1 ml/min). For enantiomer separation of the different hydroxy fatty acids, chiral-phase HPLC of the positional isomers was performed under the following conditions (flow rate, 1 ml/min): (15R/S)-HETE, Chiralcel OD column, n-hexane;–2-propanol;–acetic acid 100:5:0.1 (v/v/v); (8R/S)-HETE, Chiralcel OD column, n-hexane;–2-propanol;–acetic acid 100:4:0.1 (v/v/v); (5R/S)-HETE, OB column, n-hexane;–2-propanol;–acetic acid 100:5:0.1 (v/v/v). For 15-HETE the free acids were analyzed, and for 8- and 5-HETE the methyl esters were analyzed.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IL-4 induces 15-LO-I gene expression in HUVEC
Fig 1A shows the effects of IL-4 on 15-LO-I gene expression in HUVEC, using a semiquantitative RT-PCR technique. No 15-LO-I mRNA was detected in cells that were cultured in the absence of IL-4. However, in HUVEC cultured for various time periods in the presence of 10 ng of IL-4 per ml, 15-LO-I mRNA had significantly increased already after 4 h and continued to rise for up to 24 h. A concentration-dependent upregulation of the human 15-LO-I gene was also observed in IL-4-treated HUVEC ( Fig 1B). Maximal activation of the 15-LO-I gene was detected in cells exposed to 50 ng of IL-4 per ml. When we compared the levels of 15-LO-I mRNA in IL-4-treated HUVEC and IL-4-treated A549 cells we found them to be similar (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 1. Time- and concentration-dependent upregulation of 15-LO-I mRNA expression in human endothelial cells by IL-4. Total cDNA was synthesized from 1 µg of cellular RNA isolated from HUVEC stimulated in a culture with (A) IL-4 at 10 ng/ml for 4, 12, and 24 h or (B) IL-4 at concentrations of 10, 25, and 50 ng/ml for 24 h. PCR products were analyzed by 2% agarose gel electrophoresis and visualized by phosphoimaging technology. The predicted sizes of RT-PCR products for 15-LO-I and ß-actin (arrows) are 952 and 188 bp, respectively. M, Molecular weight markers (100-bp DNA ladder). C: The effect of cycloheximide on 15-LO-I mRNA expression in human endothelial cells by IL-4. Cells were pretreated for 2 h with cycloheximide (CHX) at 10 µg/ml and stimulated in culture with IL-4 at 10 ng/ml for an additional 4 h. Total cDNA was synthesized and RT-PCR products were analyzed as described in (A). CTL, Control.

To determine whether IL-4-induced expression of the 15-LO-I gene requires de novo synthesis of additional transcription factors, experiments were performed in which HUVEC were pretreated with cycloheximide (10 µg/ml) for 2 h before exposure to IL-4 (10 ng/ml). As shown in Fig 1C, cycloheximide, a potent inhibitor of protein synthesis, did not affect IL-4-stimulated 15-LO-I gene expression.

IL-4-induced transcription of the 15-LO-I gene is not paralleled by a concomitant production of functional 15-LO-I protein
To investigate whether IL-4-induced expression of 15-LO-I mRNA is paralleled by a concomitant production of the functional enzyme, we measured the 15-LO-I activity of IL-4-treated cells in the presence of exogenous substrate. Surprisingly, we were unable to detect the formation of specific LO products. Although hydroxy fatty acids were formed during incubation of the HUVEC with exogenous polyenoic fatty acids (1.57 ng of HETE per mg cellular protein without IL-4 treatment, 3.3 ± 1.1 ng of HETE per mg cellular protein at an IL-4 concentration of 10 ng/ml, 2.9 ± 0.2 ng of HETE per mg cellular protein at an IL-4 concentration of 50 ng/ml), more detailed structural analysis suggested a nonlipoxygenase origin. In fact, the 15-HETE isolated from IL-4-treated cells was largely racemic. It should be stressed at this point that we detected large amounts of 15-LO-I mRNA in these cells. The negative activity assays were confirmed by Western blot experiments (data not shown). Here again, we were unable to detect 15-LO-I protein under different experimental conditions (24-, 36-, and 48-h incubations; IL-4 at 10 and 50 ng/ml).

The lack of functional 15-LO-I protein in the presence of the corresponding mRNA in IL-4-treated HUVEC suggested that elements of translational regulation may be involved in the expression of 15-LO-I. In contrast, for IL-4-treated A549 cells an increase in 15-LO-I mRNA and functional enzyme protein has previously been reported (17). For site-by-site comparison, we treated A549 cells and HUVEC with IL-4 and analyzed the expression of both 15-LO-I mRNA and 15-LO-I activity. As shown in Table 1, comparable amounts of 15-LO-I mRNA were detected in both cell types after 48 h of incubation. In contrast, only IL-4-treated A549 cells catalyzed the formation of chiral (15S)-HETE from arachidonic acid. These data indicate that expression of 15-LO-I mRNA is induced in both cell types at a comparable degree, but mRNA translation appeared to be inhibited in HUVEC. As a possible reason for the translational inhibition of the 15-LO-I mRNA, the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) K and/or hnRNP E1 at the 3'-untranslated region of the mRNA has been discussed (12). We carried out Western blot experiments with HUVEC, using a polyclonal antibody against hnRNP E1, and observed a strongly positive band at the expected molecular weight (data not shown). These data indicate that hnRNP E1 is expressed in HUVEC, but it remains to be determined whether this protein is responsible for the inability of human vascular endothelial cells to express a functional 15-LO-I. Work is in progress in our laboratories to address this question.

IL-4 activates the binding of transcription factors to the 5'-flanking region of the 15-LO-1 gene
To elucidate the mechanisms of IL-4-induced transcription of the 15-LO-I gene, the effects of this cytokine on the promoter-binding activity of STAT6 ( Fig 2), AP-2 ( Fig 3), GATA-1 ( Fig 4), NF-1 ( Fig 5), and SP-1 ( Fig 6) were examined. Putative binding sites for these transcription factors exist in the 5'-flanking region of the human 15-LO-I gene. Electrophoretic mobility shift assays indicated that IL-4 activates the binding of all transcription factors tested. A low endogenous binding activity of AP-2, GATA-1, and SP-1 was observed in nonstimulated HUVEC ( Fig 3A, Fig 4A, and Fig 6A, respectively). Maximal binding of AP-2, GATA-1, NF-1, and SP-1 was found after a 2-h exposure to IL-4 ( Fig 3 Fig 4 Fig 5 Fig 6, respectively) and a decline of the binding signal was measured after longer incubation periods. For STAT6 the maximal binding activity was already reached after a 0.5-h exposure to IL-4 and the signal was less pronounced after 2 and 24 h of exposure ( Fig 2). Concentration-dependent effects of IL-4 on transcription factor binding were observed for AP-2 and SP-1 after a 0.5-h IL-4 treatment ( Fig 3A and Fig 6A, respectively) as well as for STAT6 and GATA-1 after a 2-h exposure to IL-4 ( Fig 2A and Fig 4A, respectively).

View larger version (62K): [in this window] [in a new window]   Figure 2. The effect of IL-4 treatment on STAT6 DNA-binding activity in human endothelial cells. HUVEC were either untreated or treated with IL-4 (10, 25, and 50 ng/ml) for the indicated period of times. Nuclear extracts were prepared and analyzed by EMSA. Competition studies were performed by the addition of excess unlabeled oligonucleotide (A). Supershift analysis of STAT6-binding activity is shown in (B). Nuclear extracts were prepared from HUVEC treated for 0.5 h with IL-4 (50 ng/ml) and incubated with anti-STAT6 antibody (2, 10, and 20 ng) for 25 min before the addition of [32P]-labeled probe. Arrows indicate the specific STAT6-DNA binding, and SS represents supershift band.

View larger version (63K): [in this window] [in a new window]   Figure 3. The effect of IL-4 treatment on AP-2 DNA-binding activity in human endothelial cells. Experiments were performed as indicated in the legend to Fig 2. The effect of anti-AP-2 antibody on AP-2-binding activity is shown in (B). Nuclear extracts were prepared from HUVEC treated for 0.5 h with IL-4 (50 ng/ml) and incubated with anti-AP-2 antibody (1, 2, and 4 µg) for 25 min before the addition of [32P]-labeled probe. Arrows indicate the specific AP-2-DNA binding.

View larger version (72K): [in this window] [in a new window]   Figure 4. The effect of IL-4 treatment on GATA-1 DNA-binding activity in human endothelial cells. Experiments were performed as indicated in the legend to Fig 2. The effect of anti-GATA-1 antibody on GATA-1-binding activity is shown in (B). Nuclear extracts were prepared from HUVEC treated for 0.5 h with IL-4 (50 ng/ml) and incubated with anti-GATA-1 antibody (1, 2, and 4 µg) for 25 min before the addition of [32P]-labeled probe. Arrows indicate the specific GATA-1-DNA binding.

View larger version (68K): [in this window] [in a new window]   Figure 5. The effect of IL-4 treatment on NF-1 DNA-binding activity in human endothelial cells. Experiments were performed as indicated in the legend to Fig 2. The effect of anti-NF-1 antibody on NF-1-binding activity is shown in (B). Nuclear extracts were prepared from HUVEC treated for 0.5 h with IL-4 (50 ng/ml) and incubated with anti-NF-1 antibody (2, 5, and 10 µg) for 25 min before the addition of [32P]-labeled probe. Arrows indicate the specific NF-1-DNA binding.

View larger version (68K): [in this window] [in a new window]   Figure 6. The effect of IL-4 treatment on SP-1 DNA-binding activity in human endothelial cells. Experiments were performed as indicated in the legend to Fig 2. Supershift analysis of SP-1-binding activity is shown in (B). Nuclear extracts were prepared from HUVEC treated for 0.5 h with IL-4 (50 ng/ml) and incubated with anti-SP-1 antibody (20, 100, and 200 ng) for 25 min before the addition of [32P]-labeled probe. Arrows indicate the specific SP-1-DNA binding, and SS represents supershift band.

The identities of the EMSA bands were confirmed by three different experimental approaches: i) competition binding with the molar excess of unlabeled probes, ii) binding with mutant oligonucleotides, and iii) supershift with antibodies against studied transcription factors. As shown in Fig 2A Fig 3 Fig 4 Fig 5 Fig 6 (last lane), a molar excess of unlabeled oligonucleotide probe completely wiped out bands that corresponded to IL-4-induced activation of the individual transcription factors. In addition, nuclear extracts isolated from IL-4-treated HUVEC did not bind to the labeled mutant oligonucleotide ( Fig 2B Fig 3 Fig 4 Fig 5 Fig 6). In experiments with antibodies against individual transcription factors, two different patterns of changes were observed. Supershifts of antigen-antibody complexes were observed in studies of IL-4-induced activation of STAT6 and SP-1 ( Fig 2B and Fig 6B, respectively). In contrast, antibodies against the remaining transcription factors diminished the intensity of bands corresponding to activated transcription factors ( Fig 3B and Fig 5B).

In addition to STAT6, AP-2, GATA-1, NF-1, and SP-1, the effects of IL-4 on activation of nuclear factor B (NF-B) and activator protein 1 (AP-1) were studied in HUVEC. NF-B- and AP-1-binding sites are not represented in the promoter region of the human 15-LO-I gene. Therefore, these transcription factors served as negative controls in the present study. Exposure to IL-4 for up to 2 h did not activate NF-B and AP-1 in HUVEC (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Evidence indicates that 15-LO-I may play a significant role in the development of atherosclerosis (2) (3). Although this enzyme is not expressed in normal vessels, it can be detected in foamy macrophages of atherosclerotic lesions (31). The detection of specific peroxidation products in the lesional lipids indicated the in vivo functionality of 15-LO-I (32) (33). Interestingly, the precursor cells of foamy macrophages, that is, the peripheral monocytes, do not express 15-LO-I. Therefore, it may be concluded that 15-LO-I expression is induced during monocyte/macrophage transition and/or during foam cell formation.

In this study, we investigated the ability of IL-4 to upregulate expression of 15-LO-I in human vascular endothelial cells. RT-PCR with primer combinations specific for the human reticulocyte-type 15-LO-I indicated the induction of 15-LO-I in HUVEC in a dose- and time-dependent manner. Restriction mapping and sequencing of the final PCR products confirmed the identity of the 15-LO-I band (data not shown). Although the 15-LO-I mRNA was consistently induced in IL-4-treated HUVEC, this induction was not associated with the production of functional 15-LO-I protein. These data suggested additional regulatory elements in 15-LO-I expression in HUVEC. It has been reported previously that in young rabbit reticulocytes no 15-LO-I is synthesized, although large amounts of 15-LO-I mRNA are present (12). In these cells, translation of 15-LO-I mRNA is prevented by the binding of specific nuclear proteins (hnRNP K and hnRNP E1) to a repetitive sequence motif in the 3'-untranslated region of the 15-LO-I mRNA (11) (12). Because hnRNP E1 is expressed in HUVEC (data not shown), similar mechanisms may be responsible for the lack of 15-LO-I protein expression in HUVEC.

In the past, several reports have demonstrated the occurrence of 15-HETE and 13-hydroxyoctadecadienoic acid (13-HODE) in different types of endothelial cells including HUVEC (34) (35) (36) (37) (38) (39) (40). In fact, on the basis of these observations, it was suggested that endothelial cells may exhibit 15-LO-I activity (40). It should, however, be stressed that the detection of 15-HETE does not prove the expression of 15-LO-I. 15-HETE and 13-HODE can also be formed via other metabolic pathways. Cyclooxygenases 1 and 2, which are expressed in endothelial cells (41) (42) (43), are capable of forming 13-HODE under certain experimental conditions and, thus, may contribute to hydroxy fatty acid synthesis. Furthermore, the inhibition of HETE/HODE formation by eicosa-5,8,11,14-tetraynoic acid or nordihydroguaiaretic acid is not convincing, because both compounds lack LO specificity (44) (45). Thus, for the time being there is no compelling evidence for the expression of 15-LO-I in vascular endothelial cells. The results obtained in this study indicate the absence of functional 15-LO-I in IL-4-treated HUVEC in the presence of the corresponding mRNA. These data suggest a translational control of 15-LO-I expression. It may, however, be possible that under certain experimental conditions the translational control is overcome and a functional 15-LO-I might be expressed. Unfortunately, at present the mechanisms of translational control in HUVEC have not been investigated and the experimental conditions under which these processes may be downregulated have not yet been defined.

Although HUVEC are unable to translate the 15-LO-I mRNA, transcription of the 15-LO-I gene is strongly upregulated in response to IL-4. To understand the mechanism of this process, we first investigated the time course of 15-LO transcription. As indicated in Fig 1A, we observed a significant increase in 15-LO-I mRNA expression after a relatively short exposure period (4 h). Such a quick response is consistent with the hypothesis that 15-LO-I may belong to the family of immediate-early genes of the IL-4 response in HUVEC. This notion is also supported by our cycloheximide experiments. In contrast, in IL-4-treated monocytes, much longer incubation periods were necessary for effective 15-LO-I induction (13). In fact, in monocytes, IL-13 apparently induces the expression of a novel protein, which is capable of binding to the 15-LO-I promoter and, thus, may act as transcription factor in 15-LO-I expression (46).

The unexpected time course of IL-4-induced 15-LO-I gene transcription prompted us to study the activation pattern of several transcription factors for which specific binding sites have previously been identified in the 5'-flanking region of the 15-LO-I gene (47). Our data indicate that IL-4 treatment of HUVEC activates promoter binding of STAT6, AP-2, GATA-1, NF-1, and SP-1. The biological roles of these transcription factors have previously been investigated in other experimental systems. Many activities of IL-4 might be mediated by the activation of STAT proteins. For example, IL-4 can specifically activate STAT6 binding (48) in hematopoietic cells. Palmer-Crocker, Hughes, and Pober (49) and Lugli et al. (50) reported that IL-4 and IL-13 activate the Janus kinase 2 (JAK2) tyrosine kinase and STAT6 in HUVEC, and in monocyte/macrophages both STAT6 and JAK2 kinases appear to be involved in IL-4-induced upregulation of 15-LO-I (19) (20). Transcription factors of the AP-2 family, which appear to be instrumental in the development of ectodermally derived tissues, specifically bind to the DNA consensus sequence CCCCAGGC to initiate transcription of selected genes (51) (52). GATA-1 is an erythroid-related transcription factor that is involved in the generation of the erythroid lineage (53). NF-1 constitutes a family of CCAAT box-binding proteins that stimulate DNA replication and active transcription. NF-1 can bind to its consensus DNA element as a homodimer via an amino-terminal DNA-binding domain. In addition, it can activate transcription through a proline-rich, carboxy-terminal trans-activation domain (54). NF-1 recognizes and binds the adenovirus type 2 promoter and activates transcription of herpes simplex virus thymidine kinase genes (55). SP-1 is a sequence-specific transcription factor that recognizes GGGGCGGGGC and closely related sequences, which are often referred to as GC boxes. It was initially identified as a HeLa cell-derived factor that selectively activates in vitro transcription from the simian virus 40 (SV40) promoter and binds to the multiple GC boxes in the 21-bp repeated elements in SV40 (56). SP-1 belongs to a subgroup of transcription factors that are phosphorylated on binding to promoter sequences (57). However, the detailed roles of STAT6, AP-2, GATA-1, NF-1, and SP-1 in IL-4-induced expression of the 15-LO-I gene in human endothelial cells remain yet to be identified.

	ACKNOWLEDGMENTS

This work was supported in part by the Alexander von Humboldt Foundation and by research grants from the Deutsche Forschungsgemeinschaft, American Heart Association, United States Department of Agriculture, United States Department of Defense, and National Institutes of Health.

Manuscript received May 4, 2000; and in revised form December 20, 2000

Abbreviations: EMSA, electrophoretic mobility shift assay; 15-HETE, 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; hnRNP, heterogeneous nuclear ribonucleoprotein; 15-HPETE, 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; (13S)-HPODE, (13S)-hydroperoxy-9Z,11E-octadecadienoic acid; HUVEC, human umbilical vein endothelial cells; IL-4, interleukin 4; 15-LO-I, reticulocyte-type 15-lipoxygenase; RT-PCR, reverse transcriptase-polymerase chain reaction; Th2, helper T cell type 2

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Ueda, N., H. Suzuki, and S. Yamamoto. 1998. Mammalian lipoxygenases: structure, function, and evolutionary aspects. In Eicosanoids and Related Compounds in Plants and Animals. A. F. Rowley, H. Kühn, and T. Schewe, editors. Portland Press, London.

2. Feinmark, S. J., Cornicelli, J. A. 1997. Is there a role for 15-lipoxygenase in atherogenesis? Biochem. Pharmacol. 54:953-959.

3. Kühn, H., Chan, L. 1997. The role of 15-lipoxygenase in atherogenesis: pro- and antiatherogenic actions. Curr. Opin. Lipidol. 8:111-117.

4. Sparrow, C. P., Parthasarathy, S., Steinberg, D. 1998. Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A₂ mimics cell-mediated oxidative modification. J. Lipid Res. 29:745-753.

5. Belkner, J., Stender, H., Kühn, H. 1998. The rabbit 15-lipoxygenase preferentially oxygenates LDL cholesterol esters, and this reaction does not require vitamin E. J. Biol. Chem. 273:23225-23232.

6. Ylä-Herttuala, S., Rosenfeld, M. E., Parthasarathy, S., Glass, C. K., Sigal, E., Witztum, J. T., Steinberg, D. 1990. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc. Natl. Acad. Sci. USA. 87:6959-6963.

7. Sendobry, S. M., Cornicelli, J. A., Welch, K., Bocan, T., Tait, B., Trivedi, B. K., Colbry, N., Dyer, R. D., Feinmark, S. J., Daugherty, A. 1997. Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties. Br. J. Pharmacol. 120:1199-1206.

8. Cyrus, T., Witztum, J. L., Rader, D. J., Tangirala, R., Fazio, S., Linton, M. R. F., Funk, C. D. 1999. Disruption of the 12/15-LO-IX gene diminishes atherosclerosis in apolipoprotein E deficient mice. J. Clin. Invest. 103:1597-1604.

9. Kühn, H., Heydeck, D., Brinckman, R., Trebus, F. 1999. Regulation of cellular 15-lipoxygenase activity on pre-translational, translational and post-translational levels. Lipids. 34:S273-S279.

10. Thiele, B. J., Andree, H., Höhne, M., Rapoport, S. M. 1982. Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational depression. Eur. J. Biochem. 129:133-141.

11. Ostareck-Lederer, A., Ostareck, D. H., Standart, N., Thiele, B. J. 1994. Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region. EMBO J. 13:1476-1481.

12. Ostareck, D. H., Ostareck-Lederer, A., Wilm, M., Thiele, B. J., Mann, M., Hentze, M. W. 1997. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end. Cell. 89:597-606.

13. Conrad, D. J., Kühn, H., Mulkins, M., Highland, E., Sigal, E. 1992. Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase. Proc. Natl. Acad. Sci. USA. 89:217-221.

14. Levy, B. D., Romano, M., Chapman, H. A., Reilly, J. J., Drazen, J., Serhan, C. N. 1993. Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-trans-eicosatetraenoic acid and lipoxins. J. Clin. Invest. 92:1572-1579.

15. Kamitani, H., Geller, M., Eling, T. 1998. Expression of 15-lipoxygenase by human colorectal carcinoma Caco-2 cells during apoptosis and cell differentiation. J. Biol. Chem. 273:21569-21577.

16. Profita, M., Vignola, A. M., Sala, A., Mirabella, A., Siena, L., Pace, E., Folco, G., Bonsignore, G. 1999. Interleukin-4 enhances 15-lipoxygenase activity and incorporation of 15(S)-HETE into cellular phospholipids in cultured pulmonary epithelial cells. Am. J. Respir. Cell Mol. Biol. 20:61-68.

17. Brinckmann, R., Topp, M. S., Zalan, I., Heydeck, D., Ludwig, P., Kühn, H., Berdel, W. E., Habenicht, A. J. R. 1996. Regulation of 15-lipoxygenase expression in lung epithelial cells by interleukin-4. Biochem. J. 318:305-312.

18. Nassar, G. M., Morrow, J. D., Roberts, L. J., Lakkis, F. G., Badr, K. F. 1994. Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes. J. Biol. Chem. 269:27631-27634.

19. Heydeck, D., Thomas, L., Schnurr, K., Trebus, F., Thierfelder, W. E., Ihle, J. N., Kühn, H. 1998. Interleukin-4 and -13 induce upregulation of the murine macrophage 12/15-lipoxygenase activity: evidence for the involvement of transcription factor STAT6. Blood. 92:2503-2510.

20. Roy, B., Cathcart, M. K. 1998. Induction of 15-lipoxygenase expression by IL-13 requires tyrosine phosphorylation of Jak2 and Tyk2 in human monocytes. J. Biol. Chem. 273:32023-32029.

21. Moser, R., Groscurth, P., Carballido, J. M., Bruijnzeel, P. L., Blaser, K., Heusser, C. H., Fehr, J. 1993. Interleukin-4 induces tissue eosinophilia in mice: correlation with its in vitro capacity to stimulate the endothelial cell-dependent selective transmigration of human eosinophils. J. Lab. Clin. Med. 122:567-575.

22. Paul, W. E. 1991. Interleukin-4: a prototypic immunoregulatory lymphokine. Blood. 77:1859-1870.

23. Rocken, M., Racke, M., Shevach, E. M. 1996. IL-4-induced immune deviation as antigen-specific therapy for inflammatory autoimmune disease. Immunol. Today. 17:225-231.

24. Nonaka, M., Nonaka, R., Woolley, K., Adelroth, E., Miura, K., Okhawara, Y., Glibetic, M., Nakano, K., O'Byrne, P., Dolovich, J. 1995. Distinct immunohistochemical localization of IL-4 in human inflamed airway tissues. IL-4 is localized to eosinophils in vivo and is released by peripheral blood eosinophils. J. Immunol. 155:3234-3244.

25. Sasaguri, T., Arima, N., Tanimoto, A., Shimajiri, S., Hamada, T., Sasaguri, Y. 1998. A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells. Atherosclerosis. 138:247-253.

26. Barks, J. L., McQuillan, J. J., Iademarco, M. F. 1997. TNF-alpha and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells. J. Immunol. 159:4532-4538.

27. Young, V. M., Toborek, M., Yang, F., McClain, C. J., Hennig, B. 1998. Effect of linoleic acid on endothelial cell inflammatory mediators. Metabolism. 47:566-572.

28. Beg, A. A., Finco, T. S., Nantermet, P. V., Baldwin, A. S., Jr. 1993. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol. Cell. Biol. 13:3301-3310.

29. Toborek, M., Garrido, R., Malecki, A., Kaiser, S., Mattson, M. P., Hennig, B., Young, B. 2000. Nicotine attenuates arachidonic acid-induced overexpression of nitric oxide synthase in cultured spinal cord neurons. Exp. Neurol. 161:609-620.

30. Lees, M. B., Paxman, S. 1972. Modification of the Lowry procedure for the analysis of proteolipid protein. Anal. Biochem. 47:184-192.

31. Hiltunen, T., Luoma, J., Nikkari, T., Ylä-Herttuala, S. 1995. Induction of 15-lipoxygenase mRNA and protein in early atherosclerotic lesions. Circulation. 92:3297-3303.

32. Kühn, H., Belkner, J., Zaiss, S., Fahrenklemper, T., Wohlfeil, S. 1994. Involvement of 15-lipoxygenase in early stages of atherogenesis. J. Exp. Med. 179:1903-1911.

33. Folcik, V. A., Nivar-Aristy, R. A., Krajewski, L. P., Cathcart, M. K. 1995. Lipoxygenase contributes to the oxidation of lipids in human atherosclerotic plaques. J. Clin. Invest. 96:504-510.

34. Hopkins, N. K., Oglesby, T. D., Bundy, G. L., Gorman, R. R. 1984. Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells. J. Biol. Chem. 259:14048-14053.

35. Revtyak, G. E., Johnson, A. R., Campbell, W. B. 1988. Cultured bovine coronary arterial endothelial cells synthesize HETEs and prostacyclin. Am. J. Physiol. 254:C8-C19.

36. Honda, H. M., Leitinger, N., Frankel, M., Goldhaber, J. I., Natarajan, R., Nadler, J. L., Weiss, J. N., Berliner, J. A. 1999. Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites. Arterioscler. Thromb. Vasc. Biol. 19:680-686.

37. Patricia, M. K., Kim, J. A., Harper, C. M., Shih, P. T., Berliner, J. A., Natarajan, R., Nadler, J. L., Hedrick, C. C. 1999. Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells. Arterioscler. Thromb. Vasc. Biol. 19:2615-2622.

38. Camacho, M., Godessart, N., Anton, R., Garcia, M., Vila, L. 1995. Interleukin-1 enhances the ability of cultured human umbilical vein endothelial cells to oxidize linoleic acid. J. Biol. Chem. 270:17279-17286.

39. Parthasarathy, S., Wieland, E., Steinberg, D. 1989. A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein. Proc. Natl. Acad. Sci. USA. 86:1046-1050.

40. Buchanan, M. R., Haas, T. A., Lagarde, M., Guichardant, M. 1985. 13-Hydroxyoctadecadienoic acid is the vessel wall chemorepellant factor, LOX. J. Biol. Chem. 260:16056-16059.

41. Jackson, B. A., Goldstein, R. H., Roy, R., Cozzani, M., Taylor, L., Polgar, P. 1993. Effects of transforming growth factor beta and interleukin-1 beta on expression of cyclooxygenase 1 and 2 and phospholipase A2 mRNA in lung fibroblasts and endothelial cells in culture. Biochem. Biophys. Res. Commun. 197:1465-1474.

42. Akarasereenont, P., Mitchell, J. A., Bakhle, Y. S., Thiemermann, C., Vane, J. R. 1995. Comparison of the induction of cyclooxygenase and nitric oxide synthase by endotoxin in endothelial cells and macrophages. Eur. J. Pharmacol. 273:121-128.

43. Crofford, L. J. 1997. COX-1 and COX-2 tissue expression: implications and predictions. J. Rheumatol. 24(Suppl. 49):15-19.

44. Lopez, S., Vila, L., Breviario, F., de Castellarnau, C. 1993. Interleukin-1 increases 15-hydroxyeicosatetraenoic acid formation in cultured human endothelial cells. Biochim. Biophys. Acta. 1170:17-24.

45. Kalgutkar, A. S., Crews, B. C., Rowlinson, S. W., Marnett, A. B., Kozak, K. R., Remmel, R. P., Marnett, L. J. 2000. Biochemically based design of cyclooxygenase-2 (COX-2) inhibitors: facile conversion of nonsteroidal antiinflammatory drugs to potent and highly selective COX-2 inhibitors. Proc. Natl. Acad. Sci. USA. 97:925-930.

46. Kelavkar, U., Wang, S., Montero, A., Murtagh, J., Shah, K., Badr, K. 1998. Human 15-lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes. Mol. Biol. Rep. 25:173-182.

47. Kritzik, M. R., Ziober, A. F., Dicharry, S., Conrad, D. J., Sigal, E. 1997. Characterization and sequence of an additional 15-lipoxygenase transcript and of the human gene. Biochim. Biophys. Acta. 1352:267-281.

48. Schindler, C., Darnell, J. E., Jr. 1995. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621-651.

49. Palmer-Crocker, R. L., Hughes, C. C. W., Pober, J. S. 1996. IL-4 and IL-13 activate the JAK2 tyrosine kinase and Stat6 in cultured human vascular endothelial cells through a common pathway that does not involve the _c chain. J. Clin. Invest. 98:604-609.

50. Lugli, S. M., Feng, N., Heim, M. H., Adam, M., Schnyder, B., Etter, H., Yamage, M., Eugster, H. P., Lutz, R. A., Zurawski, G., Moser, R. 1997. Tumor necrosis factor enhances the expression of the interleukin (IL)-4 receptor ;–chain on endothelial cells increasing IL-4 or IL-13-induced Stat6 activation. J. Biol. Chem. 272:5487-5494.

51. Williams, T., Admon, A., Luscher, B., Tjian, R. 1998. Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes Dev. 2:1557-1569.

52. Moser, M., Imhof, A., Pscherer, A., Bauer, R., Amselgruber, W., Sinowatz, F., Hofstadter, F., Schule, R., Buettner, R. 1995. Cloning and characterization of a second AP-2 transcription factor: AP-2ß. Development. 12:2779-2788.

53. Ko, L. J., Engel, J. D. 1993. DNA-binding specificities of the GATA transcription factor family. Mol. Cell. Biol. 13:4011-4022.

54. Mermod, N., O'Neill, E. A., Kelly, T. J., Tjian, R. 1989. The proline-rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA binding domain. Cell. 58:741-753.

55. Santoro, C., Mermod, N., Andrews, P. C., Tjian, R. 1988. A family of human CCAAT-box binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature. 334:218-224.

56. Dynan, W. S., Tjian, R. 1983. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 35:79-87.

57. Jackson, S. P., MacDonald, J. J., Lees-Miller, S., Tjian, R. 1990. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell. 63:155-165.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. H. Wei, Y. Yang, G. Wu, and L. J. Ignarro IL-4 and IL-13 upregulate ornithine decarboxylase expression by PI3K and MAP kinase pathways in vascular smooth muscle cells Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1198 - C1205. [Abstract] [Full Text] [PDF]

				N. T. Aggarwal, Y. Chawengsub, K. M. Gauthier, H. Viita, S. Yla-Herttuala, and W. B. Campbell Endothelial 15-Lipoxygenase-1 Overexpression Increases Acetylcholine-Induced Hypotension and Vasorelaxation in Rabbits Hypertension, February 1, 2008; 51(2): 246 - 251. [Abstract] [Full Text] [PDF]

				V. L. King, L. A. Cassis, and A. Daugherty Interleukin-4 Does Not Influence Development of Hypercholesterolemia or Angiotensin II-Induced Atherosclerotic Lesions in Mice Am. J. Pathol., December 1, 2007; 171(6): 2040 - 2047. [Abstract] [Full Text] [PDF]

				B. Chen, S. Tsui, W. E. Boeglin, R. S. Douglas, A. R. Brash, and T. J. Smith Interleukin-4 Induces 15-Lipoxygenase-1 Expression in Human Orbital Fibroblasts from Patients with Graves Disease: EVIDENCE FOR ANATOMIC SITE-SELECTIVE ACTIONS OF Th2 CYTOKINES J. Biol. Chem., July 7, 2006; 281(27): 18296 - 18306. [Abstract] [Full Text] [PDF]

				X. Tang, B. B. Holmes, K. Nithipatikom, C. J. Hillard, H. Kuhn, and W. B. Campbell Reticulocyte 15-Lipoxygenase-I Is Important in Acetylcholine-Induced Endothelium-Dependent Vasorelaxation in Rabbit Aorta Arterioscler. Thromb. Vasc. Biol., January 1, 2006; 26(1): 78 - 84. [Abstract] [Full Text] [PDF]

				S G Baidya and Q-T Zeng Helper T cells and atherosclerosis: the cytokine web Postgrad. Med. J., December 1, 2005; 81(962): 746 - 752. [Abstract] [Full Text] [PDF]

				R. Natarajan and J. L. Nadler Lipid Inflammatory Mediators in Diabetic Vascular Disease Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1542 - 1548. [Abstract] [Full Text] [PDF]

				T. Minami, A. Sugiyama, S.-Q. Wu, R. Abid, T. Kodama, and W. C. Aird Thrombin and Phenotypic Modulation of the Endothelium Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 41 - 53. [Abstract] [Full Text] [PDF]

				X. Tang, N. Spitzbarth, H. Kuhn, P. Chaitidis, and W. B. Campbell Interleukin-13 Upregulates Vasodilatory 15-Lipoxygenase Eicosanoids in Rabbit Aorta Arterioscler. Thromb. Vasc. Biol., October 1, 2003; 23(10): 1768 - 1774. [Abstract] [Full Text] [PDF]

				J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]

				T. Minami, Md. R. Abid, J. Zhang, G. King, T. Kodama, and W. C. Aird Thrombin Stimulation of Vascular Adhesion Molecule-1 in Endothelial Cells Is Mediated by Protein Kinase C (PKC)-delta -NF-kappa B and PKC-zeta -GATA Signaling Pathways J. Biol. Chem., February 21, 2003; 278(9): 6976 - 6984. [Abstract] [Full Text] [PDF]

				Y. W. Lee, B. Hennig, and M. Toborek Redox-regulated mechanisms of IL-4-induced MCP-1 expression in human vascular endothelial cells Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H185 - H192. [Abstract] [Full Text] [PDF]

				T. Minami and W. C. Aird Thrombin Stimulation of the Vascular Cell Adhesion Molecule-1 Promoter in Endothelial Cells Is Mediated by Tandem Nuclear Factor-kappa B and GATA Motifs J. Biol. Chem., December 7, 2001; 276(50): 47632 - 47641. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles