(Circulation. 1995;92:3297-3303.)
© 1995 American Heart Association, Inc.
Articles |
Induction of 15-Lipoxygenase mRNA and Protein in Early Atherosclerotic Lesions
From the Department of Medical Biochemistry, Medical School, University of Tampere (T.H., T.N.); the Department of Clinical Chemistry, Tampere University Hospital (T.H.); and the A.I. Virtanen Institute (J.L., S.Y.-H.) and the Department of Medicine (S.Y.-H.), University of Kuopio, Finland.
Correspondence to Dr Seppo Ylä-Herttuala, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, Fin-70211 Kuopio, Finland.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background 15-Lipoxygenase (15-LO) may be involved in atherogenesis and in oxidative modification of LDL. In this study, we investigated 15-LO expression in developing atherosclerotic lesions and verified the exact type of the atherosclerosis-associated LO at the nucleotide level.
Methods and Results Quantitative reverse transcription–polymerase chain reaction, in situ hybridization, and immunocytochemistry were used in two models of experimental atherosclerosis. New Zealand White rabbits were given a 1% cholesterol diet for 0 (control group), 3, 6, or 14 weeks. 15-LO mRNA was undetectable in the aortic intima-medias of the control group, whereas it was clearly induced as early as after 3 weeks. 15-LO expression increased further in the 6- and 14-week groups. According to in situ hybridization and immunocytochemical studies, 15-LO was localized to macrophage-rich areas. In Watanabe heritable hyperlipidemic rabbits, 15-LO mRNA was undetectable in normal aortic intima-medias. 15-LO mRNA was markedly induced in fatty streaks but less so in more advanced lesions. Identification of the induced LO as reticulocyte-type 15-LO was done by cloning and sequencing. We also observed a distinct basal expression of copper-zinc and extracellular superoxide dismutases in normal aortic intima-medias, but no clear induction of these mRNAs was detected in atherosclerotic aortas.
Conclusions The results show that, in contrast to copper-zinc and extracellular superoxide dismutases, the expression of reticulocyte-type 15-LO is markedly induced in rabbit fatty streaks. This may lead to an increase in the oxidative potential during the early phase of atherogenesis and contribute to the development of atherosclerotic lesions.
Key Words: atherosclerosis • polymerase chain reaction • 15-lipoxygenase • superoxide dismutase
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Oxidative modification of LDL plays an important role in atherogenesis.1 In vivo, several mechanisms may lead to LDL oxidation. These include 15-LO, hydroxyl radical, superoxide anion, peroxynitrite, myeloperoxidase, and ceruloplasmin (for review see Reference 2). In more advanced lesions, autooxidation may contribute to the peroxidation of lesion lipids.3 4
LOs catalyze dioxygenation of fatty acids containing a 1,4-cis,cis-pentadiene structure. Their nomenclature is based on the predominantly oxygenated carbon of free arachidonic acid, but some LOs can also use other substrates.5 Several lines of evidence suggest that LOs are involved in LDL oxidation. Soybean 15-LO,6 recombinant human reticulocyte 15-LO,7 and LO reaction products8 can cause LDL oxidation in vitro. On the other hand, various LO inhibitors block cell-mediated oxidation of LDL.9 10 Reticulocyte-type 15-LO is expressed in human and rabbit atherosclerotic lesions.11 12 LO reaction products have been detected in atherosclerotic lesions,13 14 and transfer of 15-LO cDNA into fibroblasts leads to enhanced levels of lipid hydroperoxides in LDL.15 In addition, transfer of 15-LO gene into rabbit iliac arteries leads to the appearance of oxidation-specific lipid-protein adducts in the transduced arteries.16 However, verification of the lesion-associated LO type at the nucleotide level has not been reported. Little is also known about the time course and regulation of 15-LO expression during the early phase of atherogenesis.
Several antioxidative mechanisms protect cells and LDL from oxidative damage.2 3 These include various antioxidative enzymes, such as CuZn-SOD, EC-SOD, and Mn-SOD, glutathione peroxidases, and catalase.3 CuZn-SOD is mainly a cytoplasmic enzyme. Mn-SOD is located in mitochondria, whereas EC-SOD functions principally at cell membranes and in extracellular space.17 To obtain an estimate about the expression of antioxidative enzymes in developing atherosclerotic lesions, we analyzed the expression of CuZn-SOD and EC-SOD in the same samples that were used for the 15-LO analyses.
RT-PCR has substantially improved the detection of gene transcripts in experimental models in which only limited amounts of tissue are available for analyses. Competitive RT-PCR is based on the simultaneous amplification of a target gene and an internal standard that have identical primer binding sites.18 19 20 In this study, we used competitive RT-PCR to quantify the expression of 15-LO, CuZn-SOD, and EC-SOD mRNAs at various phases of atherogenesis. The 15-LO findings were confirmed with in situ hybridization and immunocytochemistry. The results suggest that, in early atherosclerotic lesions, a clear increase occurs in the expression of reticulocyte-type 15-LO mRNA and protein, whereas the expression of CuZn-SOD and EC-SOD mRNAs remains unchanged.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Animals
Male NZW and WHHL rabbits were used for the studies. The NZW rabbits were 3 months old at the beginning of the experiment. They consumed either a standard diet (control rabbits; n=2) or a diet containing 1% cholesterol (wt/wt) for 3, 6, or 14 weeks (n=5 in each group). To further verify the level of 15-LO mRNA expression in aortic intima-medias of control NZW rabbits, five additional control animals were used for the analysis of 15-LO mRNA expression in normal aortas. One rabbit in the 14-week group ceased eating before the end of the experiment and was excluded from the study. WHHL rabbits were maintained on a standard diet, and 2 of them were killed at the age of 3 months, 1 at 7 months, and 1 at 18 months. In addition, 7- and 11-month-old WHHL rabbits were perfusion-fixed for in situ hybridization studies. Blood samples were collected and analyzed for plasma cholesterol content by enzymatic methods (Boehringer Mannheim). The mean plasma cholesterol concentrations of control and cholesterol-fed NZW rabbits were 1.3±0.8 and 50.5±13.8 mmol/L, respectively. WHHL rabbits had plasma cholesterol levels of 23.6±11.3 mmol/L.
The rabbits were
anesthetized with an
intravenous injection of phentanyl-fluanisone (0.3
mL/kg, Hypnorm, Jansen Pharmaceuticals) and midazolam (1 mg/kg,
Dormicum, Hoffmann-La Roche). Aortas were opened, and the extent of
atherosclerosis was recorded by naked-eye
evaluation. Aortas were classified, according to the extent of
macroscopic atherosclerotic lesions, into the following categories:
normal intima-medias, fatty streaks, and more advanced fatty
plaques. NZW rabbit controls and the 3-week group had no visible
atherosclerotic lesions. After 6 weeks on cholesterol
feeding, 
20% of the surface area of the aortic arch was covered
with fatty streaks. Rabbits in the 14-week group had extensive fatty
streaks and plaques covering almost the entire surface area of the
aortic arch. Thus, RT-PCR samples (see below) from control and 3-week
groups were macroscopically normal, whereas samples from 6- and 14-week
groups represented an average involvement of fatty streaks
and/or fatty plaques after the indicated lengths of
cholesterol feeding. To minimize the possibility of early
fatty streak formation undetectable by naked-eye examination,
macroscopically normal intima-medias were obtained from thoracic
aortas (excluding orifices of intercostal arteries) of the two young,
3-month-old WHHL rabbits. Excluded parts of the thoracic aortas and
the entire aortic arches that were covered with fatty streaks were
pooled and analyzed separately. More advanced fatty plaques
were obtained from arches and thoracic aortas of the 7- and
18-month-old WHHL rabbits. Intima-medias were dissected free of
the adventitia and stored in liquid nitrogen until isolation of RNA.
All animal studies were approved by the Experimental Animal Committee
of the University of Tampere.
RNA Isolation and RT-PCR Conditions
Total RNA was isolated by
a guanidinium thiocyanate
method.21 On average, 34±11 µg total RNA was obtained
per 100 mg (wet wt) tissue. The quality of the RNAs was checked on
agarose gel electrophoresis, which showed intact ribosomal RNA bands.
All RNA preparations were found to be free of DNA contamination by
amplifying the samples with all PCR primer pairs used in this
study.
RNA (3.5 µg per reaction) was reverse-transcribed to
first-strand cDNA with avian myeloblastosis virus reverse
transcriptase (5 U per reaction) and random hexamer primers (1 µg per
reaction) (cDNA Cycle Kit, Invitrogen). Usually, 1/200 of the
reverse-transcribed cDNA was used for a single PCR. With 15-LO, the
amount of cDNA was doubled. Negative controls were included in every
assay as previously described.22 All RT reagents were
tested to be free of any DNA contamination with the primers used in
this study. PCRs were performed in a 15-µL volume containing 50
mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.8), 0.1% Triton X-100, 1 µmol/L
each of the gene-specific primers, and 0.30 U (0.36 U for actin)
Dynazyme DNA polymerase (Finnzymes) per reaction. The
Table
lists gene-specific dNTP and
Mg2+ concentrations, annealing temperatures, and the
numbers of PCR cycles. dATP, dCTP, dGTP, and dUTP were used, except for
15-LO amplifications, in which dTTP was substituted for dUTP for better
sensitivity. The denaturation step was 3 minutes at 94°C in the first
cycle and 1 minute thereafter. The annealing step was 1 minute at the
temperature indicated in the Table. The extension step was 1
minute at
72°C, except for the last cycle (11 minutes). For quantification, 1.5
µCi of [
-33P]dCTP (2000 Ci/mmol, Du Pont NEN)
was
used per reaction in competitive PCR. Because only the internal
standard for actin was not homologous with the corresponding cDNA
fragment, there was a possibility for heterodimer formation during the
late PCR cycles.18 Consequently, except for actin, the
final cycle was performed after 1:4 dilution of the amplified sample to
bring PCR back into its exponential phase.18
|
PCR primer
sequences and lengths of the amplified fragments are given
in the Table
. Actin primers were designed according to rabbit
-smooth muscle and ß-nonmuscle actin cDNA
sequences.23 GAPDH primers were based on rabbit cDNA
sequence.24 15-LO primers were designed according to
rabbit reticulocyte 15-LO cDNA sequence,25 and they
avoided all known 5- and 12-LO sequences. CuZn-SOD primers were
designed according to human and rabbit cDNA
sequences.26 27
RT-PCR was used to clone a partial cDNA sequence of rabbit EC-SOD. Rabbit aortic RNA was subjected to RT-PCR with human primers spanning a 375-bp fragment of the human cDNA (nucleotides 171 through 545).17 The amplified fragment was isolated, cloned according to standard procedures (TA Cloning Kit, Invitrogen and pGEM-T, Promega), and sequenced from both ends with an Autoread DNA Sequencing Kit (Pharmacia P-L Biochemicals).28 The sequence between the primers has been submitted to EMBL Data Library (accession number X78139). Rabbit EC-SOD cDNA sequence shows an 86% homology with the corresponding human sequence. EC-SOD primers were designed according to the sequence information and spanned a 221-bp fragment of human and rabbit cDNAs.
Internal Standards and Competitive PCR
The same sets of
primers were used to amplify both the
internal PCR standards and the target cDNAs (Table). The
standard for
actin analyses was made from a nonhomologous DNA
(BamHI/EcoRI fragment of the v-erb B
gene, PCR Mimic Construction Kit, Clontech). The CuZn-SOD standard was
prepared by amplifying human aortic cDNA with the CuZn-SOD primers. The
human-derived standard has a Dde I (Boehringer
Mannheim) restriction site that is missing from the rabbit
amplification product. The EC-SOD standard was prepared in the same
way as the CuZn-SOD standard, but it contained a human-specific
restriction site for Sma I (Boehringer Mannheim).
The GAPDH and 15-LO standards were generous gifts from Dr H. Lukhaup
and Dr H.A. Dresel. The GAPDH standard was initially derived from
rabbit cDNA but was manipulated to have a 102-bp deletion between the
primer binding sites. The 15-LO standard was prepared analogously, and
it had a 117-bp deletion between the primer binding sites.
For quantification of gene transcripts, an equal amount of cDNA was pipetted to each of the PCRs, but different amounts of internal standard were added.20 Quantification was started with an 8- or 10-fold dilution series of the PCR standard to get an initial approximation of the cDNA concentration. After that, a twofold dilution series of the standard was made, and competitive PCR was performed with the radioactive label. However, because no clear induction of CuZn-SOD or EC-SOD mRNA expression was detected during atherogenesis, their cDNA concentrations were determined according to the intensities of the target and standard bands in ethidium bromide–stained gels (SigmaScan/Image Software, Jandel Scientific).18
The PCR products were separated (4 V/cm, 2 hours) in 2% MetaPhor agarose (FMC BioProducts) in 45 mmol/L Tris-borate buffer (pH 8.0) containing 1 mmol/L EDTA. The reactions were performed in duplicate or triplicate, and two thirds of the reactions were used for the electrophoresis. The cDNA- and standard-derived fragments were cut out from the gel, and their radioactivities were determined (1210 Ultrobeta liquid scintillation counter, LKB/Wallac). The ratio of standard versus cDNA radioactivity was counted after the background was subtracted and was further corrected according to the dCTP content of the PCR products. The ratio was plotted against the amount of the added standard. A regression line was drawn through the plot, and the amount of cDNA in the samples was deduced from the point at which the ratio of the radioactivities was 1. The cDNA concentrations were further corrected according to the lengths of the amplified fragments. The mean interassay coefficients of variation for the quantification analyses were as follows: actin 15%, GAPDH 10%, 15-LO 16%, CuZn-SOD 11%, and EC-SOD 22%.
Immunocytochemistry and In Situ Hybridization
Tissue samples
for immunocytochemistry and in situ hybridization
were taken in a standardized way from aortas of comparable animals at
the same age. Aortic tissue was rapidly removed and immersion-fixed
for 6 hours in 4% paraformaldehyde.11
Serial paraffin- or OCT-embedded tissue sections (10 µm) were used
for the analyses. 15-LO antisense and sense riboprobes were
synthesized in the presence of 35S-UTP (Du Pont NEN) from a
pBluescript SK plasmid (Stratagene) as described.11 In
situ hybridizations were performed on pretreated tissue sections
(6x106 cpm/mL). Final wash was with 0.1xSSC at
60°C for 30 minutes. Autoradiography (NTB-2,
Eastman-Kodak) was used for signal detection. Nonhybridizing sense
probes were used as controls.11
The following antibodies were used for the immunostainings: mouse mAb against macrophages (RAM-11; dilution 1:500)29 ; mouse mAb against smooth muscle cells (HHF-35; dilution 1:500)30 ; and goat antiserum against human reticulocyte–type 15-LO (dilution 1:1000), which recognizes rabbit 15-LO but does not crossreact with 5-LO or platelet-type 12-LO.11 Avidin–biotin–horseradish peroxidase system (Vector Laboratories) was used for the signal detection.11 Class- and species-matched immunoglobulins were used as controls for the immunostainings together with incubations in which the primary antibodies were omitted.11 12
Statistical Analysis
All data are expressed as
mean±SD. Quantitative RT-PCR results
were first analyzed by one-way ANOVA. After that,
Student's t test was used to compare the NZW rabbit control
and WHHL rabbit normal groups with the other groups. Values of
P<.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Actin and GAPDH mRNA Expression
An example of actin quantification in normal NZW rabbit aorta is shown in Fig 1A
. The target cDNA and the internal
standard are approximately equally amplified in the middle lane of the
insert in Fig 1A. After additional parallel
amplifications, a quantification plot was established. Similarly, the
GAPDH target cDNA from WHHL rabbit fatty streaks and the internal
standard are about equally amplified in the middle lane of the insert
in Fig 1B. The visual estimation was confirmed by a
quantification plot.
|
Our results indicate that a twofold to fivefold
induction occurs in the
amount of GAPDH mRNA in both rabbit models with the development of
atherosclerotic lesions (Fig 2). Because
actin mRNA levels remained more stable during atherogenesis, actin
expression was used to normalize the expression of other genes.
|
15-LO mRNA Expression
15-LO mRNA expression was barely
detectable in the normal NZW
aortas (Fig 2A). Similar results were obtained from
normal intima-medias of five additional control NZW rabbits, in
which 15-LO expression was also below the detection limit (ie,
<3x10-4 amol/µg total RNA). After 3
weeks on the cholesterol-rich diet, there was a 15-fold
increase in the amount of 15-LO mRNA in NZW rabbit aortas (Figs
1C and 2A). After 14 weeks on the
cholesterol-rich diet, the
expression of 15-LO mRNA was increased more than 100-fold compared with
that of the control aorta (Fig 2A).
In normal WHHL
rabbit aortas, 15-LO mRNA expression was below the
detection limit, whereas fatty streaks showed a remarkable induction of
15-LO mRNA expression (Fig 2B). When
atherosclerosis had progressed further to more advanced
fatty plaques, the content of 15-LO mRNA was 10 times lower than in the
fatty streaks.
Verification of 15-LO PCR Product
Although 15-LO PCRs were
performed under stringent
conditions (Table) and the primers were designed to avoid other
known
LOs, the identities of PCR amplification products were confirmed by
cloning and sequencing. Five insert-containing clones were
sequenced from both NZW and WHHL rabbit lesions. They all were
identical to the published sequence of rabbit reticulocyte 15-LO
cDNA,25 except for a constant G-to-A substitution at
nucleotide 607, which thus represents an allelic
variation or a true sequencing error in the original sequence.
In Situ Hybridization and Immunocytochemistry
Strong
expression of 15-LO mRNA was detected in both WHHL and NZW
rabbit atherosclerotic arteries. Fig 3a shows an example
of 15-LO mRNA expression in WHHL rabbit lesions. As a control, a serial
section was hybridized with a nonhybridizing 15-LO sense probe (Fig
3b). Immunostainings of serial sections
with mAb RAM-11 revealed that most of the cells in 15-LO–expressing
areas were macrophages (data not shown). An example of 15-LO
expression in early NZW rabbit atherosclerotic lesions is shown in Fig
3c through 3h: 15-LO protein (Fig 3d)
and mRNA (Fig 3e) were expressed in the same area that
immunostained for macrophages (Fig 3g). Control hybridizations
with the 15-LO sense probe
(Fig 3f) and control immunostainings
(Fig 3h) were negative. Macrophages expressing
15-LO mRNA were present in the NZW rabbit intima as early as after
3 weeks on the cholesterol-rich diet (data not shown).
Under stringent conditions, no 15-LO mRNA or protein was detected in
medial smooth muscle cells. However, we cannot fully exclude a
possibility for a low-level expression of 15-LO–like molecules in
arterial smooth muscle cells.
|
CuZn-SOD and EC-SOD mRNA Expressions
CuZn-SOD and EC-SOD
expressions were analyzed with
competitive RT-PCR from the same samples that were used for the 15-LO
quantification. There was a clear basal expression of both CuZn-SOD and
EC-SOD mRNA in normal aortas. However, no major changes were detected
in the expression of these genes with the progression of
atherosclerosis (Fig 2).
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
RT-PCR was chosen for the analysis of arterial wall gene expression instead of Northern blotting because of its better sensitivity. The usefulness of RT-PCR is clearly illustrated by the fact that, on average,
240 µg total RNA (or 10
µg poly[A] RNA) is obtained from aortic arch intima-medias of
five rabbits, which permits only a few Northern blotting experiments
but is still sufficient for more than 10 000 PCRs. Various strategies
for semiquantitative PCR can be used. First, PCR can be performed
without any added standard, but it is very difficult to standardize the
assay without coamplification of a standard molecule. It is also
possible to add to the PCR tube another set of primers designed to
amplify an unrelated standard gene (such as actin). However, the
standard gene is usually expressed in higher amounts, and its
amplification has already reached a plateau when the gene of interest
is barely detectable. In addition, multiple primer sets in the same
tube can interfere with the amplification process.31 We
used a competitive PCR technique with added standards that can be
amplified in the same tube with the same set of primers as the target
cDNA.18 19 20 This approach eliminates
most of the problems
related to the above-mentioned amplification methods and can be
used to study differences in the amounts of gene transcripts between
various samples. The standard may have either a homologous or a
heterologous sequence between the PCR primers, since amplification
efficiency seems to be determined primarily by the primer
sequences.19 31
Initially, we aimed to use two genes, actin and GAPDH, for the
normalization of the results. However, a twofold to fivefold induction
of GAPDH mRNA expression was detected in both rabbit models with the
development of atherosclerotic lesions (Fig 2). The
increase in GAPDH expression may be due to an increasing number of
macrophages in the arterial wall during progression
of atherosclerosis, to a change in smooth muscle cells
from a contractile to a synthetic phenotype,32 or
to the effect of high blood cholesterol content on
glycolytic metabolism in the cells. Additionally, nuclear
uracil DNA glycosylase is a product of the same gene as
GAPDH,33 which brings on another source of variation in
GAPDH mRNA expression. These data suggest that GAPDH should not be used
for the normalization of mRNA expression at different phases of
atherogenesis. Conversely, only random changes were detected in actin
expression between the samples. Consequently, actin was chosen as the
primary gene for the normalization of the quantification results.
Nevertheless, no qualitative changes in the 15-LO quantification
results would have been caused by the use of GAPDH as a normalizing
gene; only the magnitude of 15-LO induction would have been smaller
(Fig 2).
No 15-LO activity measurements were performed in the present study. However, our results are in close agreement with those of Kühn et al,14 who measured 15-LO reaction products in NZW rabbit atherosclerotic arteries using a study design very similar to our study. In their study, the amount of the S-enantiomer of 13-HODE, compared with the R-enantiomer, was slightly increased after 6 weeks and substantially increased after 14 weeks on a 1% cholesterol diet, which indicates increased enzymatic oxidation of linoleic acid. Thus, their results imply an induction of 15-LO expression in early atherosclerotic lesions, which was found in the present study.
Our results show that 15-LO mRNA expression is induced in NZW rabbit
aortas after as little as 3 weeks on a cholesterol-rich
diet before the appearance of the first macroscopic lesions. At this
stage, some macrophages were already present in the
arterial wall. The concentration of 15-LO mRNA was highest
in NZW rabbit aortas after 14 weeks on the
cholesterol-rich diet and in aortic fatty streaks of
3-month-old WHHL rabbits. On the basis of in situ hybridization and
immunocytochemical studies, it is likely that most of the 15-LO
expression occurs in macrophages. This study confirms previous
findings11 12 of the 15-LO mRNA and protein
expression in
lesion macrophages. When atherosclerosis in
WHHL rabbits had further progressed to more advanced fatty plaques, the
15-LO mRNA levels had fallen to levels about 1/10 of the levels found
in the fatty streaks (Fig 2). Results of Kühn et
al14 also suggest a decreased role for enzymatic (15-LO)
oxidation in the later phase of atherogenesis, in which it is more
likely that unspecific lipid peroxidation plays an important role.
Reduction of 15-LO expression in the advanced lesions may be due to a
lower number of macrophages or to a downregulation of 15-LO
expression and/or macrophage activity by various
cytokines present in atherosclerotic
lesions.34 We did not detect any major induction of
CuZn-SOD or EC-SOD mRNA in rabbit atherosclerotic aortas. Thus, a
definite increase in 15-LO versus SOD mRNA ratio was observed during
the early phase of atherogenesis.
Previous reports of 15-LO expression in atherosclerotic lesions were based on qualitative RT-PCR, in situ hybridization, immunoblotting, immunocytochemistry,11 12 and the demonstration of the presence of 15-LO reaction products in atherosclerotic aortas.13 14 In the former studies, leukocyte-type 5-LO and platelet-type 12-LO mRNA could not be detected in the lesion area.11 12 However, a possibility of cross-reaction between members of the same gene family exists. Also, detection of 13-HODE does not unequivocally prove 15-LO function, since leukocyte-type 12-LO converts linoleic acid to 13-HODE.35 36 37 Consequently, we cloned and sequenced the atherosclerosis-associated LO. The results confirmed the induction of the reticulocyte-type 15-LO expression in rabbit atherosclerotic lesions. The present results do not exclude the possibility of an additional expression of a leukocyte-type 12-LO or some unknown LOs in lesion macrophages, endothelial cells, or smooth muscle cells. However, the results suggest that 15-LO expression in macrophages may play an important role in the early phase of atherogenesis and could be a potential target for interventions.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by grants from The Elli and Elvi Oksanen Fund of the Pirkanmaa Regional Fund under the auspices of the Finnish Cultural Foundation, The Academy of Finland, Sigrid Jusélius Foundation, Finnish Heart Foundation, and Aarne Koskelo Foundation. The authors thank Drs Joseph L. Witztum, Daniel Steinberg, and Elliott Sigal for providing reticulocyte 15-LO antibodies and cDNA, Dr Allen Gown for RAM-11 and HHF-35 antibodies, and Drs Heike Lukhaup and Hans A. Dresel for GAPDH and 15-LO standards. Eija Kyrölä's expertise in typing the manuscript is acknowledged.
Received March 7, 1995; revision received July 11, 1995; accepted July 16, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785-1792.
2. Ylä-Herttuala S. Role of lipid and lipoprotein oxidation in the pathogenesis of atherosclerosis. Drugs Today. 1994;30:507-514.
3. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. Oxford, England: Clarendon Press; 1989.
4. Smith C, Mitchinson MJ, Aruoma OI, Halliwell B. Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions. Biochem J. 1992;286:901-905.
5. Yamamoto S. Mammalian lipoxygenases: molecular structures and functions. Biochim Biophys Acta. 1992;1128:117-131. [Medline] [Order article via Infotrieve]
6. Sparrow CP, Parthasarathy S, Steinberg D. Enzymatic modification of low density lipoprotein by purified lipoxygenase and phospholipase A2 mimics cell-mediated oxidative modification. J Lipid Res. 1988;29:745-753. [Abstract]
7. Belkner J, Wiesner R, Rathman J, Barnett J, Sigal E, Kühn H. Oxygenation of lipoproteins by mammalian lipoxygenases. Eur J Biochem. 1993;213:251-261. [Medline] [Order article via Infotrieve]
8. O'Learly VJ, Darley-Usmar VM, Russell LJ, Stone D. Pro-oxidant effects of lipoxygenase-derived peroxides on the copper-initiated oxidation of low-density lipoprotein. Biochem J. 1992;282:631-634.
9.
Parthasarathy S, Wieland E, Steinberg D. A role
for endothelial cell lipoxygenase
in the oxidative modification of low density lipoprotein.
Proc Natl Acad Sci U S A. 1989;86:1046-1050.
10. Rankin SM, Parthasarathy S, Steinberg D. Evidence for a dominant role of lipoxygenase(s) in the oxidation of LDL by mouse peritoneal macrophages. J Lipid Res. 1991;32:449-456. [Abstract]
11.
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.
12. 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.
13. Simon TC, Makheja AN, Bailey JM. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) as the predominant eicosanoid in aortas from Watanabe heritable hyperlipidemic and cholesterol-fed rabbits. Atherosclerosis. 1989;75:31-38. [Medline] [Order article via Infotrieve]
14.
Kühn H, Belkner J, Zaiss S, Fährenklemper
T, Wohlfeil S. Involvement of
15-lipoxygenase in early stages of
atherogenesis. J Exp Med. 1994;179:1903-1911.
15.
Benz DJ, Mol M, Ezaki M, Mori-Ito N, Zalán I,
Miyanohara A, Friedmann T, Parthasarathy S, Steinberg D, Witztum
JL. Enhanced levels of lipoperoxides in low density lipoprotein
incubated with murine fibroblasts expressing high levels of human
15-lipoxygenase. J Biol
Chem. 1995;270:5191-5197.
16. Ylä-Herttuala S, Luoma J, Viita H, Hiltunen T, Sisto T, Nikkari T. Transfer of 15-lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein. J Clin Invest. 1995;95:2692-2698.
17.
Hjalmarsson K, Marklund SL, Engström Å, Edlund
T. Isolation and sequence of complementary DNA encoding human
extracellular superoxide dismutase. Proc Natl Acad Sci
U S A. 1987;84:6340-6344.
18.
Becker-André M, Hahlbrock K. Absolute mRNA
quantification using the polymerase chain reaction (PCR): a novel
approach by a PCR aided transcript titration assay (PATTY).
Nucleic Acids Res. 1989;17:9437-9446.
19.
Wang AM, Doyle MV, Mark DF: Quantitation of mRNA by the
polymerase chain reaction. Proc Natl Acad Sci U S A. 1989;86:9717-9721.
20.
Gilliland G, Perrin S, Blanchard K, Bunn HF.
Analysis of cytokine mRNA and DNA: detection and
quantitation by competitive polymerase chain reaction.
Proc Natl Acad Sci U S A. 1990;87:2725-2729.
21. Kingston RE. CsCl purification of RNA from tissue. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York, NY: Greene Publishing Assocs and John Wiley & Sons; 1991:4.2.3-4.2.4.
22. Hiltunen T, Raja-Honkala M, Nikkari T, Ylä-Herttuala S. A PCR artifact under low-stringency conditions due to amplification by only one primer. Biotechniques. 1994;17:240-242. [Medline] [Order article via Infotrieve]
23.
Harris DE, Warshaw DM, Periasamy M.
Nucleotide sequence of the rabbit -smooth-muscle
and ß-non-muscle actin mRNAs. Gene. 1992;112:265-266. [Medline]
[Order article via Infotrieve]
24. Applequist S, Keyna U, Beck-Engeser G, Raman C, Jack HM. Nucleotide sequence of rabbit glyceraldehyde-3-phosphate dehydrogenase mRNA. EMBL Data Library 1993: Accession No. L23961. European Bioinformatics Institute, Hinxton Genome Campus, Cambridge CB10 1RQ, England. E-mail datalib@ebi.ac.uk.
25. Fleming J, Thiele BJ, Chester J, O'Prey J, Janetzki S, Aitken A, Anton IA, Rapoport SM, Harrison PR. The complete sequence of the rabbit erythroid cell-specific 15-lipoxygenase mRNA: comparison of the predicted amino acid sequence of the erythrocyte lipoxygenase with other lipoxygenases. Gene. 1989;79:181-188. [Medline] [Order article via Infotrieve]
26.
Sherman L, Dafni N, Lieman-Hurwitz J, Groner Y.
Nucleotide sequence and expression of human chromosome
21-encoded superoxide dismutase mRNA. Proc Natl Acad Sci
U S A. 1983;80:5465-5469.
27. Jackson RM, Ho YS. Nucleotide and deduced amino acid sequence of rabbit CuZnSOD. EMBL Data Library 1993: Accession No. L12405. European Bioinformatics Institute, Hinxton Genome Campus, Cambridge CB10 1RQ, England. E-mail datalib@ebi.ac.uk.
28.
Ansorge W, Sproat B, Stegemann J, Schwager C, Zenke
M. Automated DNA sequencing: ultrasensitive detection of
fluorescent bands during electrophoresis. Nucleic
Acids Res. 1987;15:4593-4602.
29. Tsukada T, Rosenfeld M, Ross R, Gown AM. Immunocytochemical analysis of cellular components in atherosclerotic lesions: use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis. 1986;6:601-613. [Abstract]
30. Tsukada T, Tippens D, Gordon D, Ross R, Gown AM. HHF35, a muscle-actin-specific monoclonal antibody, I: immunocytochemical and biochemical characterization. Am J Pathol. 1987;126:51-60. [Abstract]
31. Siebert PD, Larrick JW. Competitive PCR. Nature. 1992;359:557-558. [Medline] [Order article via Infotrieve]
32. Campbell GR, Campbell JH. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis. Exp Mol Pathol. 1985;42:139-162. [Medline] [Order article via Infotrieve]
33.
Meyer-Siegler K, Mauro DJ, Seal G, Wurzer J, deRiel JK,
Sirover MA. A human nuclear uracil DNA glycosylase is the 37-kDa
subunit of glyceraldehyde-3-phosphate
dehydrogenase. Proc Natl Acad Sci U S A. 1991;88:8460-8464.
34.
Conrad DJ, Kühn 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.
35.
Yokoyama C, Shinjo F, Yoshimoto T, Yamamoto S, Oates
JA, Brash AR. Arachidonate
12-lipoxygenase purified from porcine leukocytes by
immunoaffinity chromatography and its reactivity with
hydroperoxyeicosatetraenoic acids. J Biol
Chem. 1986;261:16714-16721.
36. Hada T, Hagiya H, Suzuki H, Arakawa T, Nakamura M, Matsuda S, Yoshimoto T, Yamamoto S, Azekawa T, Morita Y, Ishimura K, Kim HY. Arachidonate 12-lipoxygenase of rat pineal glands: catalytic properties and primary structure deduced from its cDNA. Biochim Biophys Acta. 1994;1211:221-228. [Medline] [Order article via Infotrieve]
37.
Chen XS, Kurre U, Jenkins NA, Copeland NG, Funk
CD. cDNA cloning, expression, mutagenesis of C-terminal
isoleucine, genomic structure, and chromosomal localizations of
murine 12-lipoxygenases. J
Biol Chem. 1994;269:13979-13987.
This article has been cited by other articles:
 |
|
 |
|
  N. T. Aggarwal, S. L. Pfister, K. M. Gauthier, Y. Chawengsub, J. E. Baker, and W. B. Campbell Chronic hypoxia enhances 15-lipoxygenase-mediated vasorelaxation in rabbit arteries Am J Physiol Heart Circ Physiol, March 1, 2009; 296(3): H678 - H688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Nikolic, M. C. Gong, J. Turk, and S. R. Post Class A Scavenger Receptor-mediated Macrophage Adhesion Requires Coupling of Calcium-independent Phospholipase A2 and 12/15-Lipoxygenase to Rac and Cdc42 Activation J. Biol. Chem., November 16, 2007; 282(46): 33405 - 33411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. OSTERUD and E. BJORKLID Role of Monocytes in Atherogenesis Physiol Rev, October 1, 2003; 83(4): 1069 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Spanbroek, R. Grabner, K. Lotzer, M. Hildner, A. Urbach, K. Ruhling, M. P. W. Moos, B. Kaiser, T. U. Cohnert, T. Wahlers, et al. Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis PNAS, February 4, 2003; 100(3): 1238 - 1243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Heydeck, J. M. Upston, H. Viita, S. Yla-Herttuala, and R. Stocker Oxidation of LDL by rabbit and human 15-lipoxygenase: prevalence of nonenzymatic reactions J. Lipid Res., July 1, 2001; 42(7): 1082 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Fang, T. L. Kaduce, and A. A. Spector 13-(S)-Hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells J. Lipid Res., April 1, 1999; 40(4): 699 - 707. [Abstract] [Full Text]
|
|
|
|
|
|
J. Belkner, H. Stender, and H. Kuhn The Rabbit 15-Lipoxygenase Preferentially Oxygenates LDL Cholesterol Esters, and This Reaction Does Not Require Vitamin E J. Biol. Chem., September 4, 1998; 273(36): 23225 - 23232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. P. Hiltunen, J. S. Luoma, T. Nikkari, and S. Yla-Herttuala Expression of LDL Receptor, VLDL Receptor, LDL Receptor–Related Protein, and Scavenger Receptor in Rabbit Atherosclerotic Lesions : Marked Induction of Scavenger Receptor and VLDL Receptor Expression During Lesion Development Circulation, March 24, 1998; 97(11): 1079 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. West and J. A. Hubbell Separation of the arterial wall from blood contact using hydrogel barriers reduces intimal thickening after balloon injury in the rat: The roles of medial and luminal factors in arterial healing PNAS, November 12, 1996; 93(23): 13188 - 13193. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||