This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, J. A. Articles by Nadler, J. L. Search for Related Content PubMed PubMed Citation Articles by Kim, J. A. Articles by Nadler, J. L.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:942-948.)
© 1995 American Heart Association, Inc.

Articles

A Leukocyte Type of 12-Lipoxygenase Is Expressed in Human Vascular and Mononuclear Cells

Evidence for Upregulation by Angiotensin II

Presented at the American Federation for Clinical Research National Meeting, April 30-May 3, 1993, Washington, DC, and published in abstract form (Clin Res. 1993;41:148.).

Jeong A. Kim; Jia-Li Gu; Rama Natarajan; Judith A. Berliner; Jerry L. Nadler

From the City of Hope National Medical Center and UCLA School of Medicine (J.A.B.), Los Angeles, Calif.

Correspondence to Jerry L. Nadler, MD, City of Hope Medical Center, 1500 E Duarte Rd, Duarte, CA 91010.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract The lipoxygenase (LO) pathway has been implicated in leading to accelerated atherosclerosis. However, the precise type of LO present in unstimulated human aortic smooth muscle cells (HSMC), endothelial cells (HAEC), and monocytes (MO) is not clear. In this study, we used a specific reverse-transcriptase polymerase chain reaction (RT-PCR) method to analyze the type of LO mRNA expressed in normal HSMC, HAEC, and MO. In all three cell types, a 333-base-pair band was seen when primers and probes specific for the leukocyte type of 12-LO were used, suggesting that a leukocyte type of 12-LO is expressed in these cell types. Western immunoblotting analysis in cultured HSMC, HAEC, and MO using a polyclonal peptide antibody to the leukocyte type of 12-LO showed a specific 72-kD band that is identical to the molecular weight of the leukocyte type of 12-LO. These results indicate that a leukocyte type of 12-LO RNA and protein are expressed in HSMC, HAEC, and MO. Further, angiotensin II upregulates 12-LO activity and expression in HSMC, supporting a role for this 12-LO pathway in human vascular disease.

Key Words: vascular smooth muscle cells • endothelial cells • atherosclerosis • hydroxyeicosatetraenoic acids • hypertension

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The three mammalian lipoxygenases (LO) are named according to the carbon position (5, 12, or 15) at which they oxygenate arachidonic acid.¹ There is increasing evidence that certain LO enzymes are involved in the pathogenesis and acceleration of atherosclerosis by inducing oxidation of LDL to its atherogenic form^{2 3} and increasing the growth or migration of smooth muscle cells.^{4 5} In addition, new evidence suggests that a 12-LO protein plays a role in mediating angiotensin II (Ang II)–induced vascular and adrenal actions.^{6 7 8} Recent studies indicate that at least two forms of 12-LO exist: The human platelet type 12-LO cloned from human erythroleukemia cells has been found primarily in platelets.^{9 10} The other, a porcine leukocyte-type 12-LO, has been isolated and cloned from porcine leukocyte cells,^{11 12} porcine pituitary cells,¹³ and bovine tracheal cells.^{14 15} We recently demonstrated the presence of a leukocyte type of 12-LO in human adrenal glomerulosa cells.¹⁶ Human 15-LO has been purified from human and rabbit reticulocytes.^{17 18} The human platelet and porcine leukocyte-type 12-LO share 65% amino acid homology.⁹ However, porcine leukocyte-type 12-LO is highly homologous to the human 15-LO (86%).¹² Recently, it has been shown that 15-LO is expressed in macrophages of human atherosclerotic lesions but not in unstimulated monocytes (MO).^{19 20 21}

In the present study, we evaluated the precise type of LO present in unstimulated human aortic smooth muscle cells (HSMC), endothelial cells (HAEC), and MO. Furthermore, since Ang II can increase the expression of 12-LO in human adrenal cells, we also evaluated the effects of Ang II on 12-LO regulation in HSMC. The results show that a 12-LO similar to that found in human adrenal glomerulosa is expressed in the normal HSMC, HAEC, and MO. Furthermore, this 12-LO is markedly upregulated by Ang II in HSMC.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cells and Cultures
HAEC and HSMC were isolated from aortic specimens obtained from the heart donors in the UCLA heart transplant program. HAEC at passages 5 through 9 and HSMC at passages 3 through 7 were used. HAEC were grown in medium 199 containing 20% fetal bovine serum (FBS) supplemented with endothelial cell growth supplement (20 mg/mL) and heparin (90 µg/mL). HAEC were identified by their typical cobblestone morphology, presence of factor VIII–related antigen, and uptake of acetylated LDL labeled with 1,1'-dioctadecyl-1-3,3,3'3'-tetramethylindo-carbocyanine perchlorate (Dil-acetyl-LDL).²² HSMC were grown in medium 199 containing 20% FBS and identified morphologically and immunohistochemically by use of HHF35, which was then visualized by a fluorescently labeled second antibody or with a biotin-streptavidin complex immunoperoxidase system.²³ MO were obtained from a large pool of healthy donors by a modification of the Recalde method.²⁴ These cells were approximately 73±8% MO.

The MO were suspended at 15x10⁶ cells/mL in RPMI 1640 medium supplemented with L-glutamine, penicillin, and streptomycin. Cells were allowed to adhere to 100-mm polystyrene tissue culture plates for 3 hours at 37°C in the presence of 5% CO₂. The nonadherent cells were removed by rinsing the plates three times with PBS. The adherent cells were incubated in 10% FBS containing RPMI medium for 36 hours in the presence or absence of interleukin (IL)-4 (400 pmol/L, R&D Systems).^{21 25}

HSMC and HAEC monolayers were washed twice with ice-cold PBS and then processed for RNA extraction or Western analysis as described below. For HETE assay, approximately 24 hours prior to an experiment, the medium was replaced with medium 199 containing 0.4% FBS and 0.2% BSA.

cDNAs
Recombinant Bluescript plasmid containing the cDNA for human reticulocyte 15-LO was kindly provided by Dr E. Sigal (Syntex Co). pUC19 plasmid containing the cDNA for porcine leukocyte 12-LO was kindly provided by Drs S. Yamamoto and T. Yoshimoto (Tokushima University, Tokushima, Japan).¹² Bluescript plasmid containing the cDNA for human platelet 12-LO was kindly provided by Prof Bengt Samuelsson (Karolinska Institute, Stockholm, Sweden).¹⁰

Oligonucleotide Primers and Probes for Polymerase Chain Reaction
ß₂-Microglobulin oligonucleotides were a kind gift of Dr Perrin White (Cornell University Medical College, New York, NY). Other oligonucleotides, including human GAPDH oligonucleotides, were synthesized on an Applied Biosystems DNA synthesizer and were purified by polyacrylamide gel electrophoresis. The sequences of oligonucleotides are listed in the Table; they were designed on the basis of known gene sequences^{10 12 26 27} and selected from regions displaying the greatest divergence between porcine 12-LO and 15-LO sequences.⁹

View this table: [in this window] [in a new window]   Table 1. Primers and Probes for Amplification and Detection

Amplification of Reverse-Transcribed RNA by the Polymerase Chain Reaction
Total RNA from cultured HSMC and HAEC and freshly isolated MO was extracted with guanidiumthiocyanate-phenol-chloroform with RNAzol (Cinna/Biotecx Laboratories International Inc) or RNA stat-60 (Tel-test B, Inc). Some RNA samples were treated with RNAse-free DNAse. Total RNA (2.5 to 3 µg) was mixed with the polymerase chain reaction (PCR) buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, and 0.001% gelatin), 200 µmol/L of each of the four deoxynucleotide triphosphates, 25 pmol each of 5'- and 3'-primers, 2 U avian myeloblastosis virus reverse transcriptase (20 U/µL; Life Sciences), and 2.5 U Taq polymerase (Perkin Elmer Cetus) in a final volume of 50 µL. In some reactions, 5 pmol each of 5'- and 3'-primer of ß₂-microglobulin or GAPDH was added as an internal standard. The samples were placed in a thermal cycler at 37°C for 8 minutes for the reverse transcription (RT) reaction to proceed. Then, conditions used for PCR were a denaturation step at 94°C for 1 minute, annealing at 50°C for 2 minutes, and extension at 72°C for 2 minutes for 20 to 40 cycles. Blank reactions with no RNA template were carried out through the RT and PCR steps. The human 15-LO cDNA, porcine leukocyte 12-LO cDNA, and human platelet 12-LO cDNA amplifications were carried out by mixing 2 to 5 ng cDNA plasmid in a 50-µL volume containing 200 µmol/L of each of the four deoxynucleotide trisphosphates, 25 pmol 5'- and 3'-primers, and 2.5 U Taq polymerase. The conditions for semiquantitative PCR were the same as described before.¹⁶

Gel Analysis and Blot Hybridization
Aliquots (20 µL) of the PCR products were subjected to electrophoresis in a 1.8% agarose gel in Tris acetate–EDTA buffer. After being stained with ethidium bromide and photographed, the gel was transferred onto a Zeta-probe membrane (Bio-Rad) by capillary blotting. The oligonucleotides used as probes were labeled at the 5'-end with [-³²P]ATP and T4 polynucleotide kinase (New England Biolabs) and hybridized with membrane overnight in 6xSSC (1xSSC contains 0.15 mol/L NaCl/0.015 mol/L sodium citrate), 0.5% nonfat dried milk, and 7% SDS at 42°C. Membranes were washed once in 6xSSC at room temperature for 15 minutes and then once at 60°C for 15 minutes. The washing conditions were worked out to distinguish the PCR products of human 15-LO from those of porcine leukocyte 12-LO.¹⁶ The filters were exposed to Kodak x-ray film (Eastman Kodak Co) with an intensifying screen at -70°C. Blots were quantified with a computerized video densitometer.

Western Immunoblotting
Cell pellets were lysed in lysis buffer containing PBS (pH 7.3), 1% Triton X-100, 1 mmol/L PMSF, 50 µmol/L leupeptin, and 0.1% SDS. Lysates were centrifuged at 10 000g for 10 minutes. An aliquot of the supernatant (cytosol) was saved for protein estimation, and the remainder was saved at -70°C for Western blot analysis.

SDS polyacrylamide gel electrophoresis (10% running gel, 4% stacking gel) was performed according to the method of Laemmli.²⁸ For Western blotting, gels were equilibrated in transfer buffer (35 mmol/L Tris base, 192 mmol/L glycine, and 20% methanol, pH 8.3) and then transferred to nitrocellulose (Hybond, Amersham), as described by Towbin et al,²⁹ in a semidry polyblot apparatus (American Bionetics, Inc) for 40 minutes. The nonspecific sites were blocked with PBS containing 10% fetal calf serum (FCS) at 4°C overnight. The membranes were then washed twice with PBST (PBS + 0.05% Tween-20) and incubated with primary antibody in PBST containing 1% BSA and 20% (vol/vol) FCS for 2 hours at room temperature. A polyclonal antibody against porcine 12-LO peptide with the sequence of amino acids 646 through 662 of the porcine leukocyte 12-LO sequence¹² was used. This antiserum was used at 1:100 dilution. In some studies, a polyclonal antibody against human 15-LO kindly provided by Dr E. Sigal (Syntex Co) was used. The washed membranes were then incubated for 1 hour with second antibody (goat anti-rabbit) conjugated with alkaline phosphatase (1:5000; Promega). Detection was either by color development using substrate mixture (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, Promega) or by chemiluminescence using CSPD substrate and the Western-Light Chemiluminescent detection system (Tropix, Inc). Nonspecific binding was evaluated with normal rabbit serum. Western blots were quantified with a computerized video densitometer (Applied Imaging; Lynx DNA Vision), and values were expressed as arbitrary absorbance units.

Measurement of 12-LO Products
These assays were performed according to previously published methods.^{6 7} Briefly, 12- and 15-HETE are extracted from supernatants and cells on C18 mini columns (Analytichem International) and measured by our validated reverse-phase gradient high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) methods. The HPLC system provides a 0.7-minute separation time between 12- and 15-HETE peaks. The 12-LO antibody used for RIA recognizes only 12-S-HETE, with less than 0.1% cross-reactivity with 12-R-HETE and 0.3% with 15-HETE.

Measurement of LO Activity in HSMC
Confluent HSMC were placed in medium plus 10% FCS 24 hours prior to the experiment. The cells were harvested, washed, suspended in 1 mL Tris-HCl buffer (25 mmol/L, pH 7.7), and then sonicated on ice. The assay mixture contained, in 1.0 mL, 800 µL enzyme (sonicate), 100 µL CaCl₂ (1.5 mmol/L), and 50 µL glutathione (0.5 mmol/L). An enzyme blank was run simultaneously. The reaction was started at 37°C with 50 µL sodium arachidonate (160 µmol/L Nu Check Prep) or 0.25 µCi [¹⁴C]linoleic acid (New England Nuclear). After 10 minutes of incubation, the reaction was stopped with 2 mL isopropanol/1.2% acetic acid followed by 2 mL chloroform. The lower organic layer was filtered and subjected to HPLC to detect HETEs using our gradient reverse-phase HPLC system.^{6 7} A 12-HETE peak was identified by UV detection at 237 nm and comigration with authentic standard (retention time, 18.3 minutes). Peak heights were quantified with a Shimazu CR5A integrator.

Data Analysis
Immunoblots and autoradiograms were analyzed with a computer-driven densitometer (Applied Imaging; Lynx DNA Vision). Data shown are representative of 2 or 3 experiments. Data generated from Ang II treatment of HSMC for 12-HETE synthesis were analyzed by ANOVA for multiple samples using a statistical package on a Macintosh computer system. Data are presented as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Expression of a Leukocyte Type of 12-LO mRNA in HAEC, HSMC, and MO
The expression of 12-LO mRNA in HAEC, HSMC, and MO was evaluated by a specific semiquantitative RT-PCR method, since the level of detection was below the sensitivity of Northern analysis. Fig 1A shows expression of leukocyte 12-LO mRNA in normal HAEC, HSMC, and MO by a method highly specific for this form of 12-LO mRNA. The appropriate 333-base-pair (bp) band was seen in all three cell types.

View larger version (38K): [in this window] [in a new window]   Figure 1. Reverse transcriptase–polymerase chain reaction analysis of leukocyte 12-lipoxygenase (LO) RNA in human aortic endothelial cells (HAEC), human aortic smooth muscle cells (HSMC), and monocytes (MO). A, RNA samples were amplified for 40 cycles with porcine leukocyte-specific 12-LO primers. Membranes were hybridized with porcine leukocyte 12-LO oligonucleotide probe. Lane 1 is a marker; lanes 2, 5, and 8 are negative controls without template; lane 3 represents total RNA from HAEC, with porcine leukocyte 12-LO primer and lane 4 with GAPDH primers; lane 6 represents total RNA from HSMC with porcine leukocyte 12-LO primers and lane 7 with GAPDH primers; lane 9 represents total RNA from MO with porcine leukocyte 12-LO primers and lane 10 with GAPDH primers; and lane 11 is a positive control using the porcine leukocyte 12-LO cDNA. B, Same RNA samples were amplified for 40 cycles with human specific 15-LO primers. Membranes were hybridized with human 15-LO oligonucleotide probe. Only the 333-base-pair (bp) product from amplification of the 15-LO cDNA (positive control) is shown. C, Ethidium bromide–stained agarose gel of A. D, Ethidium bromide–stained agarose gel of B.

Fig 1B demonstrates RT-PCR analysis of human 15-LO mRNA expression from the same RNA. These results reveal no evidence for a band characteristic of human 15-LO. In a separate experiment, RNA from HAEC, HSMC, and MO was amplified and probed for the platelet-type 12-LO RNA. No evidence for a human platelet 12-LO expression was found (data not shown).

Evidence for Selective Increase in 15-LO mRNA in MO Exposed to IL-4
Fig 2A shows expression of human 15-LO mRNA in MO by IL-4 exposure for 36 hours. However, no 15-LO mRNA was seen in fresh or cultured human MO in the absence of IL-4.

View larger version (23K): [in this window] [in a new window]   Figure 2. Increase in human 15-lipoxygenase (LO) RNA in monocytes (MO) exposed to interleukin (IL)-4. A, RNA samples were amplified for 23 cycles with GAPDH primers or 40 cycles with human 15-LO primers. Membranes were hybridized with human 15-LO oligonucleotide probe. Lane 1 is a negative control without template; lane 2 represents total RNA from MO incubated in 10% fetal bovine serum containing RPMI 1640 for 36 hours; lane 3 from MO treated with IL-4 (400 pmol/L) for 36 hours; and lane 4 from freshly isolated MO with human 15-LO primers. Lanes 5 and 6 represent same RNA samples as lane 2 and lane 3, respectively, with GAPDH primers. Lane 7 is a positive control using the human 15-LO cDNA. Ethidium bromide–stained agarose gel is shown in the left panel. B, Same RNA samples were amplified with leukocyte 12-LO primers. Membranes were hybridized with leukocyte 12-LO oligonucleotide probe. Lane 7 is a positive control using the leukocyte 12-LO cDNA. Ethidium bromide–stained gel is shown in the left panel.

Fig 2B demonstrates expression of leukocyte 12-LO mRNA in the same RNA samples as in Fig 2A. Interestingly, 12-LO mRNA expression was actually reduced with IL-4 treatment. The left panels are ethidium bromide–stained gels, and the right panels are autoradiograms.

Expression of a Leukocyte Type of 12-LO Protein in HAEC, HSMC, and MO
To investigate whether a leukocyte type of 12-LO enzyme was expressed in HAEC, HSMC, and circulating MO, the 10 000g supernatant proteins were electrophoresed and subjected to Western analysis using a polyclonal peptide antibody derived from a sequence in the porcine leukocyte type of 12-LO that is homologous to the sequence of 12-LO found in human adrenal glomerulosa. This antibody has previously been shown to lack cross-reactivity to the platelet form of 12-LO and successfully demonstrated the presence of a leukocyte-type 12-LO in human adrenal cells.¹⁶ Fig 3 demonstrates a major 72-kD band from Western analysis in HSMC, HAEC, and MO. Western analysis performed similarly using a polyclonal antibody directed against the human 15-LO protein did not demonstrate a band in the expected molecular weight from these cells (data not shown). HAEC and MO produced 12-S-HETE as reflected by HPLC and RIA analysis (HAEC 2386, MO 820 pg/10⁶ cells). Results for HSMC are detailed below.

View larger version (45K): [in this window] [in a new window]   Figure 3. Expression of leukocyte 12-lipoxygenase (LO) protein (72 kD) in normal human aortic endothelial cells (HAEC), human aortic smooth muscle cells (HSMC), and monocytes (MO). Cytosol fractions from HAEC, HSMC, and MO were electrophoresed along with authentic porcine 12-LO protein and subjected to Western immunoblotting as described under "Methods."

Therefore, HSMC, HAEC, and MO appear to express a 12-LO protein similar to the leukocyte type of 12-LO found in porcine tissues and human adrenal glomerulosa.

Effect of Ang II on 12-LO Activity and Expression in HSMC
Fig 4A shows that 5 minutes of incubation of HSMC with Ang II at the concentrations of 10^-8 and 10^-9 mol/L in serum-free medium stimulates the release of 12-HETE (control, 599±105; Ang II 10^-8 mol/L, 1467±277; Ang II 10^-9 mol/L, 1296±262 pg/mg protein). Ten-minute incubations with Ang II significantly stimulated the release of 12-HETE at the concentration of 10^-8 mol/L. Ang II also significantly increased cell-associated 12-HETE levels in HSMC (Fig 4B). In other studies, it was found that 12-HETE release in the medium in response to Ang II (10^-7 mol/L) could be reduced by the LO inhibitor cinnamyl-3,4-dihydroxy--cyanocinnamate (CDC, 10^-5 mol/L) (control, 1064±60; Ang II, 3297±178; Ang II+CDC, 1862±116 pg/mg protein, P<.0001 versus Ang II alone). Another structurally distinct 12-LO inhibitor, baicalein (10^-5 mol/L), also reduced the cell-associated increases in 12-HETE in response to Ang II (control, 9.9±0.66; Ang II, 20.6±2.46; Ang II+baicalein, 15.7±1.2 ng/mg protein, P<.01 versus Ang II alone).

View larger version (37K): [in this window] [in a new window]   Figure 4. Bar graphs. A, Effect of angiotensin II (Ang II) on 12-HETE release by human aortic smooth muscle cells (HSMC). HSMC were grown to confluency. Serum was removed, and cells were incubated in medium 199 containing 0.4% fetal bovine serum (FBS) and 0.2% BSA for 18 hours. Cells were then washed with Dulbecco's modified Eagle's medium (DMEM) and incubated for 20 minutes in DMEM containing 0.2% BSA. Ang II was added to the cells for 5 and 10 minutes at concentrations of 10-9 and 10-8 mol/L. Media were collected for HETE assay by high-performance liquid chromatography and radioimmunoassay. * P<.05 vs control, n=4. B, Effect of Ang II on cell-associated 12-HETE levels in HSMC. After supernatants were collected, cells were washed with ice-cold PBS and harvested by scraping for the assay of cell-associated HETEs as described in the "Methods" section. * P<.02 vs control, n=4.

To examine whether Ang II induces the 12-LO enzyme expression in HSMC, cells were treated with Ang II at a concentration of 2x10^-7 mol/L for 24 or 48 hours. The 12-LO protein was identified by Western immunoblotting using a specific antibody to purified leukocyte-type 12-LO or a peptide antibody derived from known sequences present in the human leukocyte type of 12-LO. A distinct band was detected with a molecular weight of nearly 72 kD, which is the reported molecular weight of the porcine leukocyte type of 12-LO (Fig 5). A 24-hour incubation of HSMC with Ang II in serum-free medium induced nearly a sevenfold increase in 12-LO protein expression (Fig 5). In other experiments, Ang II added for 48 hours also increased 12-LO expression fourfold to sevenfold (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 5. Regulation of leukocyte 12-lipoxygenase protein expression by angiotensin II (Ang II, AII in figure) in human aortic smooth muscle cells. A, Immunoblot showing regulation by Ang II. B, Bar graph representation of densitometric analysis of immunoblot in A. Cells were grown in medium 199 containing 20% fetal bovine serum (FBS) and were serum-depleted for 24 hours by placement in medium 199 containing 0.4% FBS and 0.2% bovine serum albumin. Cells were treated with Ang II at the concentration of 2x10-7 mol/L for 24 or 48 hours. Cells were washed with PBS and harvested by scraping. Cell pellets were lysed and cytosol fractions were electrophoresed as described under "Methods."

To evaluate the specific expression and regulation of 12-LO mRNA in HSMC, we used an RT-PCR assay that exclusively amplifies the leukocyte type of 12-LO. The size of the PCR-amplified fragment is 333 bp for both 12- and 15-LO. Therefore, specific conditions were used to distinguish leukocyte-type 12-LO and human 15-LO by increasing stringency and raising washing temperature to 60°C. Fig 6A shows a Southern blot analysis of RT-PCR–amplified products from HSMC that were serum-deprived for 24 hours and then treated for the indicated times with Ang II 10^-7 mol/L. In this experiment, very low basal expression of 12-LO is seen. However, in other experiments in cells from various other donors, basal 12-LO expression is detectable with PCR at 20 to 30 cycles. Ang II induces 12-LO mRNA expression starting at the 12-hour incubation time, and the maximum induction is shown at 36 hours of incubation of cells with Ang II. Fig 6B shows the ethidium bromide–stained agarose gel showing the amplification of GAPDH as an internal marker. When PCR conditions were used that were specific for either the platelet-type 12-LO or human 15-LO, no specific RNA band was detected (data not shown). Therefore, basal serum-deprived HSMC show low expression of a leukocyte-type 12-LO, which is markedly upregulated by Ang II.

View larger version (66K): [in this window] [in a new window]   Figure 6. Regulation of leukocyte 12-lipoxygenase (LO) mRNA levels by angiotensin II (Ang II) in human aortic smooth muscle cells (HSMC) by reverse transcriptase–polymerase chain reaction. A, Autoradiogram of the blot hybridized with porcine leukocyte-type 12-LO oligonucleotide probe. B, Ethidium bromide–stained agarose gel. Total RNA was extracted from cultured HSMC incubated in low-serum conditions with Ang II (2x10-7 mol/L) for the different time periods shown. RNA samples were amplified for 40 cycles with leukocyte 12-LO primers or GAPDH primers.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present results demonstrate for the first time that a 12-LO RNA and protein similar to that found in porcine leukocytes and human adrenal glomerulosa^{12 16} are also expressed in human vascular cells and circulating MO. Several approaches were used in this present investigation to support this conclusion. First, a peptide antibody derived from a sequence common to the porcine and human forms of leukocyte-type 12-LO revealed a characteristic 72-kD band in HSMC, HAEC, and MO lysates. This antibody does not cross-react with the platelet form of 12-LO but has partial cross-reactivity with human 15-LO.¹⁶ Second, a highly specific RT-PCR procedure was used to detect 12-LO mRNA in these cell types. A previous study demonstrated, using this technique, that a leukocyte type of 12-LO was the exclusive type of 12-LO seen in human adrenal glomerulosa and U937 cells.¹⁶ In the present study, a specific 333-bp amplified mRNA product was found in unstimulated HSMC, HAEC, and MO when appropriate leukocyte-type 12-LO primers and probe were used. Third, in all three cell types, the 12-LO product 12-S-HETE was formed, as reflected by HPLC and specific RIA, which recognizes only the S enantiomer of 12-HETE. The cytosol from HSMC reacted with LO to produce 12-HETE or linoleic acid to form 13-hydroxyoctadeca-9Z,11E-dienoic acid (data not shown). This reaction is characteristic of a leukocyte type of 12-LO and not the platelet 12-LO, which reacts only with arachidonic acid to produce 12-HETE.

The human 15-LO originally cloned from the reticulocyte and found in human trachea is highly homologous (86% sequence homology) to the porcine leukocyte type of 12-LO.¹² The PCR technique used here can distinguish between the leukocyte 12-LO and the 15-LO.¹⁶ The specificity of this approach was demonstrated using the 12-LO and 15-LO cDNA as templates for amplification.¹⁶ Therefore, the Southern blot hybridization using the leukocyte 12-LO probe provides the strongest evidence that the band seen reflects a 12-LO and not a 15-LO amplified product. These results are in agreement with previous studies showing no detectable 15-LO mRNA in basal or stimulated human endothelial or nonstimulated mononuclear cells.³⁰ However, 15-LO mRNA and protein have been found in macrophage-rich areas of atherosclerotic vascular lesions^{19 20} and in IL-4 stimulated MO,^{21 25} suggesting that 15-LO can play a role in advanced atherosclerotic and immune-mediated vascular disease. In the present report, we confirm the results of others^{21 25} that IL-4 can increase 15-LO mRNA expression in human MO. In contrast, the 12-LO expression in these MO is actually reduced by IL-4, suggesting that different factors can regulate human 15-LO and the leukocyte type of 12-LO.

Another major finding of the present study is that Ang II increases the activity and expression of 12-LO mRNA and protein in HSMC. Increasing evidence suggests that a 12-LO enzyme plays an important role in Ang II–induced actions in several tissues. Studies suggest that the 12-LO pathway of arachidonic acid can mediate Ang II–induced aldosterone synthesis in rat and human adrenal glomerulosa cells.^{6 7} Furthermore, recent data indicate that Ang II–induced adrenal cell proliferation is mediated at least in part by activation of a 12-LO enzyme.⁸ Additional studies in the rat have implicated the 12-LO pathway in the vasoconstrictive and renin-inhibitory actions of Ang II.^{31 32} The aorta has the capacity to produce LO products, including 12- and 15-HETE.³³ Recent data have revealed that both Ang II and high glucose can upregulate the leukocyte type of 12-LO activity and expression in cultured porcine aortic smooth muscle cells.^{34 35}

Additional studies will be needed to fully evaluate the potential implication of the increased 12-LO activity and expression by Ang II in human vessel wall. Ang II has major effects on vascular smooth muscle cell growth in vitro and in vivo.^{4 36 37 38 39} A recent report found that a relatively selective 12-LO inhibitor could completely prevent Ang II–induced hypertrophic responses in cultured porcine vascular smooth muscle cells.⁴ Furthermore, 12-HETE induced increases in protein and fibronectin content of these vascular smooth muscle cells similar to those induced by Ang II.⁴ The 12-LO pathway and its product, 12-HETE, have also been implicated in vascular smooth muscle cell migration.⁵ 12-HETE at concentrations as low as 10^-12 mol/L have been shown to lead to smooth muscle cell migration. Additional studies have demonstrated that 12-LO products can activate specific isoforms of protein kinase C and oncogenes, including ras, c-fos, and jun.^{40 41 42} Therefore, increased 12-LO activity and expression by Ang II may be a previously unrecognized mechanism for Ang II–induced hypertensive and atherosclerotic vascular disease in humans, and blockade of the 12-LO pathway may be a novel therapeutic modality to reduce Ang II–related cardiovascular disease.

The 12-LO pathway in the human vascular wall and MO may participate in other mechanisms related to the development or progression of atherosclerotic vascular disease. Recent evidence has implicated an LO pathway in oxidative modification of LDL in the vascular wall.^{19 20} It is now clear that HAEC, HSMC, or MO have the capacity to convert native LDL to minimally modified LDL, which has a greater atherosclerotic potential. Of interest are the data showing that cholesterol loading of macrophages leads primarily to increased production of 12-HETE.⁴³ A recent report has now demonstrated that both the leukocyte type of 12-LO and 15-LO can similarly oxidize lipoproteins.⁴⁴ Interestingly, this same report showed a lack of ability of the platelet 12-LO to oxidize lipoproteins.

The precise role of this 12-LO pathway in hypertensive and atherosclerotic disease in humans will require further study using specific methods to selectively inhibit this form of 12-LO. Currently, few data exist on appropriate pharmacological inhibitors that are selective for the leukocyte type of 12-LO. However, use of antisense or ribozyme methods to reduce leukocyte-type 12-LO activity should provide more definitive information as to the role of this newly defined pathway that may be relevant to human vascular disease.

	Acknowledgments

 
These studies were supported by National Institutes of Health grants RO1-DK-39721 (Dr Nadler), R29-HL-48920 (Dr Natarajan), and HL-30568 (Dr Berliner), a Grant-in-Aid from the Los Angeles Affiliate of the American Heart Association (Dr Gu), and the Laubisch Fund. Dr Kim was supported by a Postdoctoral Fellowship Award from the Los Angeles Affiliate of the American Heart Association. The authors thank Linda Lanting, Lisa Thomas, Steven Scott, and Wei Bai for their technical assistance and Elizabeth Rees for typing the manuscript.

Received November 3, 1994; accepted April 10, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Samuelsson B, Dahlen SE, Lindgren JA, Rouzer CA, Serhan CN. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science. 1987;237:1171-1176.[Abstract/Free Full Text]
2. Sparrow CP, Parthasarathy S, Steinberg D. Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A₂ mimics cell-mediated oxidative modification. J Lipid Res. 1988;29:745-753. [Abstract]
3. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]
4. Natarajan R, Gonzales N, Lanting L, Nadler JL. Role of the lipoxygenase pathway in angiotensin II–induced vascular smooth muscle cell hypertrophy. Hypertension. 1994;23(suppl I):I-142-I-147.
5. Nakao J, Ooyama T, Ito H, Chang WC, Murota S. Comparative effect of lipoxygenase products of arachidonic acid on rat aortic smooth muscle cell migration. Atherosclerosis. 1982;44:339-342. [Medline] [Order article via Infotrieve]
6. Natarajan R, Stern N, Hsueh W, Do Y, Nadler JL. Role of the lipoxygenase pathway in angiotensin II-mediated aldosterone biosynthesis in human adrenal glomerulosa cells. J Clin Endocrinol Metab. 1988;67:584-591. [Abstract]
7. Nadler JL, Natarajan R, Stern N. Specific action of the lipoxygenase pathway in mediating angiotensin II-induced aldosterone synthesis in isolated adrenal glomerulosa cells. J Clin Invest. 1987;80:1763-1769.
8. Natarajan R, Gonzales N, Hornsby PJ, Nadler JL. Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells. Endocrinology. 1992;131:1174-1180. [Abstract]
9. Funk CD, Furci L, FitzGerald GA. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase. Proc Natl Acad Sci U S A. 1990;87:5638-5642. [Abstract/Free Full Text]
10. Izumi T, Hoshiko S, Radmark O, Samuelsson B. Cloning of the cDNA for human 12-lipoxygenase. Proc Natl Acad Sci U S A. 1990;87:7477-7481. [Abstract/Free Full Text]
11. Yoshimoto T, Miyamoto Y, Ochi K, Yamamoto S. Arachidonate 12-lipoxygenase of porcine leukocyte with activity for 5-hydroxyeicosatetraenoic acid. Biochim Biophys Acta. 1982;713:638-646. [Medline] [Order article via Infotrieve]
12. Yoshimoto T, Suzuki H, Yamamoto S, Takai T, Yokoyama C, Tanabe T. Cloning and sequence analysis of the cDNA for arachidonate lipoxygenase of porcine leukocytes. Proc Natl Acad Sci U S A. 1990;87:2142-2146. [Abstract/Free Full Text]
13. Ueda N, Hiroshima A, Natsui K, Shinjo F, Yoshimoto T, Yamamoto S, Ii K, Gerozissis K, Dray F. Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary. J Biol Chem. 1990;265:2311-2316. [Abstract/Free Full Text]
14. Hansbrough JR, Takahashi Y, Ueda N, Yamamoto S, Holtzman MJ. Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial cells distinct from leukocyte and platelet forms of the enzyme. J Biol Chem. 1990;265:1771-1776. [Abstract/Free Full Text]
15. DeMarzo N, Sloane DL, Dicharry S, Highland E, Sigal E. Molecular cloning and expression of an airway 12-lipoxygenase: a hypothesis for positional specificity. Am J Physiol. 1992;262:L198-L207. [Abstract/Free Full Text]
16. Gu J, Natarajan R, Ben-Ezra J, Valente G, Scott S, Yoshimoto T, Yamamoto S, Rossi JJ, Nadler JL. Evidence that a leukocyte type of 12-lipoxygenase is expressed and regulated by angiotensin II in human adrenal glomerulosa cells. Endocrinology. 1994;134:70-77. [Abstract]
17. Rapoport SM, Schewe T, Wiesner R, Halangk W, Ludwig P, Janicke-Hohne M, Tannert C, Hiebsch C, Klatt D. The lipoxygenase of reticulocytes: purification, characterization, and biological dynamics of the lipoxygenase: its identity with respiratory inhibitors for the reticulocyte. Eur J Biochem. 1979;96:545-561. [Medline] [Order article via Infotrieve]
18. Sigal E, Grunberger D, Craik CS, Caugney GH, Nadel GA. Arachidonate 15-lipoxygenase (W-6 lipoxygenase) from human leukocytes: purification and structural homology to other mammalian lipoxygenases. J Biol Chem. 1988;263:5328-5332. [Abstract/Free Full Text]
19. Ylä-Herttuala S, Rosenfeld ME, Parthasarathy S, Glass CK, Sigal E, Witztum JL, Steinberg D. 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 U S A. 1990;87:6959-6963. [Abstract/Free Full Text]
20. Ylä-Herttuala S, Rosenfeld ME, Parthasarathy S, Sigal E, Särkioja T, Witztum JL, Steinberg D. Gene expression in macrophage-rich human atherosclerotic lesions: 15-lipoxygenase and acetyl/low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts. J Clin Invest. 1991;87:1146-1152.
21. Conrad DJ, Kuhn H, Mulkins M, Highland E, Sigal E. Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase. Proc Natl Acad Sci U S A. 1992;89:217-221. [Abstract/Free Full Text]
22. Pitas RE, Innerarity TL, Weinstein JN, Mahley RW. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy. Arteriosclerosis. 1981;1:177-185. [Abstract/Free Full Text]
23. Gown AM, Tsukada T, Ross R. Human atherosclerosis, II: immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191-207. [Abstract]
24. Fogelman AM, Elahi F, Sykes K, VanLenten BJ, Territo MC, Berliner JA. A modification of the Recalde method for the isolation of human monocytes. J Lipid Res. 1988;29:1243-1247. [Abstract]
25. Nassar GM, Morrow JD, Roberts LJ II, Lakkis FG, Badr KF. Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes. J Biol Chem. 1994;269:27631-27634. [Abstract/Free Full Text]
26. Sigal E, Craik CS, Highland E, Grunberger D, Costello LL, Dixon RAF, Nadel JA. Molecular cloning and primary structure of human 15-lipoxygenase. Biochem Biophys Res Commun. 1988;157:457-464. [Medline] [Order article via Infotrieve]
27. Tso JY, Sun X, Kao T, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde 3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985;13:2485-2502. [Abstract/Free Full Text]
28. Laemmli NK. Cleavage of structural proteins during the assembly of the head of bacteriophage T₄. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
29. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76:4350-4354. [Abstract/Free Full Text]
30. Lopez S, Vila L, Breviario F, Castellarnau C. Interleukin-1 increases 15-hydroxyeicosatetraenoic acid formation in cultured human endothelial cells. Biochim Biophys Acta. 1993;1170:17-24. [Medline] [Order article via Infotrieve]
31. Stern N, Golub M, Nozawa K, Berger M, Knoll E, Yanagawa N, Natarajan R, Nadler JL, Tuck ML. Selective inhibition of angiotensin II mediated vasoconstriction by lipoxygenase blockade. Am J Physiol. 1989;257:H434-H443. [Abstract/Free Full Text]
32. Antonopilai I, Nadler JL, Horton R. Angiotensin feedback inhibition on renin release is expressed via the lipoxygenase pathway. Endocrinology. 1988;122:1277-1281. [Abstract]
33. Funk CD, Powell WS. Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus. J Biol Chem. 1985;260:7481-7488. [Abstract/Free Full Text]
34. Natarajan R, Gu J, Rossi J, Gonzales N, Lanting L, Xu L, Nadler J. Elevated glucose and angiotensin II increase 12-lipoxygenase activity and expression in porcine aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1993;90:4947-4951. [Abstract/Free Full Text]
35. Natarajan R, Gonzales N, Xu L, Nadler JL. Vascular smooth muscle cells exhibit increased growth in response to elevated glucose. Biochem Biophys Res Commun. 1992;187:552-560. [Medline] [Order article via Infotrieve]
36. Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension. 1989;13:305-314. [Abstract/Free Full Text]
37. Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988;62:749-756. [Abstract/Free Full Text]
38. Daemen MJAP, Lombardi DM, Bosman FT, Schwartz SM. Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circ Res. 1991;68:450-456. [Abstract/Free Full Text]
39. Osterrieder W, Miller RKM, Powell JS, Chozel JP, Hefti F, Baumgartner HR. Role of Ang II in injury-induced neointima formation in rats. Hypertension. 1991;18(suppl II):II-60-II-64.
40. Haliday EM, Ramesha CS, Ringold F. TNF induces c-fos via a novel pathway requiring conversion of arachidonic acid to a lipoxygenase metabolite. EMBO J. 1991;10:109-115. [Medline] [Order article via Infotrieve]
41. Yu CL, Tsai M, Stacey DW. Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity. Mol Cell Biol. 1990;10:6683-6689. [Abstract/Free Full Text]
42. Rao GN, Lasseque B, Griendling KK, Alexander RW. Hydrogen peroxide stimulates transcription of c-jun in vascular smooth muscle cells: role of arachidonic acid. Oncogene. 1993;8:2759-2764. [Medline] [Order article via Infotrieve]
43. Mathur S, Field J, Spector A, Armstrong M. Increased production of lipoxygenase products by cholesterol-rich mouse macrophages. Biochim Biophys Acta. 1985;837:13-19. [Medline] [Order article via Infotrieve]
44. Kuhn H, Belkner J, Suzuki H, Yamamoto S. Oxidative modification of human lipoproteins by lipoxygenases of different positional specificities. J Lipid Res. 1994;35:1749-1759.[Abstract]

This article has been cited by other articles:

				S. W. Reinhold, H. Vitzthum, T. Filbeck, K. Wolf, C. Lattas, G. A. J. Riegger, A. Kurtz, and B. K. Kramer Gene expression of 5-, 12-, and 15-lipoxygenases and leukotriene receptors along the rat nephron Am J Physiol Renal Physiol, April 1, 2006; 290(4): F864 - F872. [Abstract] [Full Text] [PDF]

				A. M. Taylor, R. Hanchett, R. Natarajan, C. C. Hedrick, S. Forrest, J. L. Nadler, and C. A. McNamara The Effects of Leukocyte-Type 12/15-Lipoxygenase on Id3-Mediated Vascular Smooth Muscle Cell Growth Arterioscler. Thromb. Vasc. Biol., October 1, 2005; 25(10): 2069 - 2074. [Abstract] [Full Text] [PDF]

				Y. Huo, L. Zhao, M. C. Hyman, P. Shashkin, B. L. Harry, T. Burcin, S. B. Forlow, M. A. Stark, D. F. Smith, S. Clarke, et al. Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E-Deficient Mice Circulation, October 5, 2004; 110(14): 2024 - 2031. [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]

				Y. Chen, D. Lasaitiene, B. G. Gabrielsson, L. M.S. Carlsson, H. Billig, B. Carlsson, N. Marcussen, X.-F. Sun, and P. Friberg Neonatal Losartan Treatment Suppresses Renal Expression of Molecules Involved in Cell-Cell and Cell-Matrix Interactions J. Am. Soc. Nephrol., May 1, 2004; 15(5): 1232 - 1243. [Abstract] [Full Text] [PDF]

				A Liguori, P Abete, J.M Hayden, F Cacciatore, F Rengo, G Ambrosio, D Bonaduce, M Condorelli, P.D Reaven, and C Napoli Effect of glycaemic control and age on low-density lipoprotein susceptibility to oxidation in diabetes mellitus type 1 Eur. Heart J., November 2, 2001; 22(22): 2075 - 2084. [Abstract] [PDF]

				R. Limor, G. Weisinger, S. Gilad, E. Knoll, O. Sharon, A. Jaffe, F. Kohen, E. Berger, B. Lifschizt-Mercer, and N. Stern A Novel Form of Platelet-Type 12-Lipoxygenase mRNA in Human Vascular Smooth Muscle Cells Hypertension, October 1, 2001; 38(4): 864 - 871. [Abstract] [Full Text] [PDF]

				J.-L. Gu, H. Pei, L. Thomas, J. L. Nadler, J. J. Rossi, L. Lanting, and R. Natarajan Ribozyme-Mediated Inhibition of Rat Leukocyte-Type 12-Lipoxygenase Prevents Intimal Hyperplasia in Balloon-Injured Rat Carotid Arteries Circulation, March 13, 2001; 103(10): 1446 - 1452. [Abstract] [Full Text] [PDF]

				M. Zhu, R. Natarajan, J. L. Nadler, J. M. Moore, C. H. Gelband, and C. Sumners Angiotensin II Increases Neuronal Delayed Rectifier K+ Current: Role of 12-Lipoxygenase Metabolites of Arachidonic Acid J Neurophysiol, November 1, 2000; 84(5): 2494 - 2501. [Abstract] [Full Text] [PDF]

				O. Meilhac, M. Zhou, N. Santanam, and S. Parthasarathy Lipid peroxides induce expression of catalase in cultured vascular cells J. Lipid Res., August 1, 2000; 41(8): 1205 - 1213. [Abstract] [Full Text]

				F. Stanke-Labesque, P. Devillier, P. Bedouch, J. L. Cracowski, O. Chavanon, and G. Bessard Angiotensin II-induced contractions in human internal mammary artery: effects of cyclooxygenase and lipoxygenase inhibition Cardiovasc Res, August 1, 2000; 47(2): 376 - 383. [Abstract] [Full Text] [PDF]

				D. Nie, K. Tang, C. Diglio, and K. V. Honn Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase Blood, April 1, 2000; 95(7): 2304 - 2311. [Abstract] [Full Text] [PDF]

				M. K. Patricia, J. A. Kim, C. M. Harper, P. T. Shih, J. A. Berliner, R. Natarajan, J. L. Nadler, and C. C. Hedrick Lipoxygenase Products Increase Monocyte Adhesion to Human Aortic Endothelial Cells Arterioscler. Thromb. Vasc. Biol., November 1, 1999; 19(11): 2615 - 2622. [Abstract] [Full Text] [PDF]

				H. M. Honda, N. Leitinger, M. Frankel, J. I. Goldhaber, R. Natarajan, J. L. Nadler, J. N. Weiss, and J. A. Berliner Induction of Monocyte Binding to Endothelial Cells by MM-LDL : Role of Lipoxygenase Metabolites Arterioscler. Thromb. Vasc. Biol., March 1, 1999; 19(3): 680 - 686. [Abstract] [Full Text] [PDF]

				R. Natarajan, H. Pei, J.-L. Gu, J. M. Sarma, and J. Nadler Evidence for 12-lipoxygenase induction in the vessel wall following balloon injury Cardiovasc Res, February 1, 1999; 41(2): 489 - 499. [Abstract] [Full Text] [PDF]

				D. Heydeck, L. Thomas, K. Schnurr, F. Trebus, W. E. Thierfelder, J. N. Ihle, and H. Kuhn Interleukin-4 and -13 Induce Upregulation of the Murine Macrophage 12/15-Lipoxygenase Activity: Evidence for the Involvement of Transcription Factor STAT6 Blood, October 1, 1998; 92(7): 2503 - 2510. [Abstract] [Full Text] [PDF]

				F. Sigari, C. Lee, J. L. Witztum, and P. D. Reaven Fibroblasts That Overexpress 15-Lipoxygenase Generate Bioactive and Minimally Modified LDL Arterioscler. Thromb. Vasc. Biol., December 1, 1997; 17(12): 3639 - 3645. [Abstract] [Full Text]

				R. Natarajan, J. Rosdahl, N. Gonzales, and W. Bai Regulation of 12-Lipoxygenase by Cytokines in Vascular Smooth Muscle Cells Hypertension, October 1, 1997; 30(4): 873 - 879. [Abstract] [Full Text]