Download The 5-lipoxygenase pathway in arterial wall biology and

Biochimica et Biophysica Acta 1736 (2005) 30 – 37
http://www.elsevier.com/locate/bba
Review
The 5-lipoxygenase pathway in arterial wall biology and atherosclerosis
Katharina Lötzer a,1, Colin D. Funk b, Andreas J.R. Habenicht a,*,1
a
Institute for Vascular Medicine, Friedrich-Schiller-University of Jena, Bachstr. 18, 07743 Jena, Germany
Departments of Physiology and Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
b
Received 2 June 2005; received in revised form 5 July 2005; accepted 8 July 2005
Available online 21 July 2005
Abstract
Leukotrienes (LTs) are powerful inflammatory lipid mediators derived from the 5-lipoxygenase (5-LO) cascade of arachidonic acid.
Recent clinical, population genetic, cell biological, and mouse studies indicate participation of the 5-LO pathway in atherogenesis and arterial
wall remodeling. 5-LO is expressed by leukocytes including blood monocytes, tissue macrophages, dendritic cells, neutrophils, and mast
cells. LTB4 and the cysteinyl LTs LTC4, LTD4, and LTE4, act through two BLT and two cysLT receptors that are differentially expressed on
hematopoietic and arterial wall cells. The precise roles of LTs or the LT receptors in cardiovascular physiology remain largely to be explored.
In this review, we will discuss what is currently known about the 5-LO atherosclerosis connection. We will attempt to propose strategies to
further explore potential links between the 5-LO pathway and blood vessel physiology and disease progression.
D 2005 Elsevier B.V. All rights reserved.
Keywords: 5-Lipoxygenase; Leukotriene; Arachidonic acid; Inflammation; Cardiovascular disease; Atherogenesis
1. The 5-LO pathway of arachidonic acid metabolism
produces mediators of blood vessel homeostasis and
inflammation
LTs, i.e., LTB4 and the cysLTs (i.e. LTC4, LTD4, and
LTE4 also known as ‘‘slow reacting substances of anaphylaxis’’), constitute a group of 5-LO-derived arachidonic acid
oxidation products that exert multiple activities in the
cardiovascular (CV) system [1 – 3]. LT formation is initiated
by activation of cytosolic phospholipase A2 resulting in
arachidonic acid release from membrane glycerophospholipids [3]. Metabolism of unesterified arachidonic acid to LTs
is achieved by 5-LO together with an associated protein,
referred to as 5-LO-activating protein (FLAP) that appears
to serve as an arachidonic acid binding and transfer protein
thereby facilitating 5-LO enzyme activity. The intermediate
metabolite, i.e. LTA4, is converted either to LTB4 or to
cysLTs by the downstream enzymes LTA4 hydrolase and
* Corresponding author.
E-mail address: [email protected] (A.J.R. Habenicht).
1
www.med.uni-jena.de/ivm/.
1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2005.07.001
LTC4 synthase, respectively [3]. LTs exert biological effects
through binding to and activation of one of four G-proteincoupled putative 7 membrane domain-type cell surface
receptors (see below). Expression patterns of LT receptors
on target cells in blood vessels and atherosclerotic lesions
are likely to govern the biological responses in vivo. There
are few leukocytes in normal arteries most of which are
found in the loose connective tissue that surrounds blood
vessels, i.e., the lamina adventitia. In human diseased
arteries, the number of 5-LO+ macrophages expands during
lesion formation in patients afflicted with late stage
atherosclerosis. In clinically significant atherosclerosis,
macrophages make up a significant portion of plaques. It
seems likely that these macrophages produce LTs although
physiological agonists that trigger arachidonic acid liberation and LT biosynthesis in vivo have not yet been
identified [4]. We proposed that LTs may trigger circuits
of arterial wall inflammation and remodeling during
clinically significant stages of atherosclerosis and have
referred to this 5-LO involvement in arterial wall biology as
the 5-LO atherosclerosis hypothesis (Fig. 1). Of leukocytes
that are found in atherosclerotic lesions, macrophages, foam
cells, dendritic cells, and mast cells express 5-LO, whereas
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
31
Fig. 1. The 5-LO atherosclerosis hypothesis of arterial wall inflammation. Macrophages/foam cells/dendritic cells express cytosolic phospholipase A2 (cPLA2)
and the 5-LO cascade. cPLA2 activation hydrolyzes arachidonic acid (AA) from the sn2 position of membrane glycerophospholipids. The released unesterified
AA binds to FLAP which transfers it to 5-LO facilitating AA conversion to 5-H(P)ETE (5-hydro(pero)xyeicosatetraenoic acid) and LTA4. LTA4 serves as
substrate for LTC4 synthase to generate LTC4/LTD4 (black arrows) or for LTA4 hydrolase to generate LTB4 (red arrows). LT formation may occur in lamina
intima lesions or in the lamina adventitia. Macrophages, T cells, mast cells, SMCs, and endothelial cells express LT-Rs. CysLTs act on cysLT1-Rs on
macrophages in an autocrine (bent black arrows) or on cysLT1-R on other neighboring cells including lymphocytes and/or SMCs in a paracrine fashion (straight
black arrows). CysLTs may also activate endothelial cells in a paracrine manner (straight black arrows). Moreover, LTB4 may act on neighboring cells
including, SMCs, and T lymphocytes in a paracrine fashion (red straight arrows) and on macrophages in an autocrine fashion (red bent arrows). Inset shows an
endothelial cell and genes that have been identified by microarray analyses 60 min after stimulation of cultured human umbilical vein endothelial cells with 100
nM LTD4: CAMs (cell adhesion molecules), MIP-2 (macrophage inflammatory protein 2), IL-8 (interleukin 8). Note that the precise LT-R pattern on cells
forming inflammatory circuits in the arterial wall has not yet been determined.
T cells do not. However, all plaque-forming cells including
T cells, endothelial cells, and smooth muscle cells may
express LT receptors. Lipid mediators with prominent proinflammatory activities such as cholesterol oxidation products, lysophospholipids, ceramide, platelet-activating factor,
oxidized long chain unsaturated fatty acids, oxidized LDL,
and eicosanoids have long been proposed to contribute to
arterial wall inflammation [5] but the precise role of each
largely remains to be determined. Though recognized more
than 20 years ago by Corey and coworkers for their effects
on intact blood vessels [6], LTs have not been perceived as
promoters of CV disease (CVD). Evidence regarding a role
of the 5-LO pathway in atherogenesis has only recently
emerged from past in vitro [7 –14], morphological [4], and
pharmacological studies [15 – 20] and data from genetically
engineered mice [21,22] (see below). Most in vivo effects of
added LTs involve interaction of leukocytes and the
endothelium [17]. Actions of LTs in blood vessels include
coronary artery contraction [15,18], decline in left ventricular contractility [18], edema formation [17], blood pressure
regulation, and leukocyte recruitment into the perivascular
space [17]. LTs promote P-selectin surface expression, von
Willebrand factor secretion, and platelet-activating factor
synthesis in cultured endothelial cells [7,11,13], and
stimulate arterial smooth muscle cell proliferation in vitro
[12]. Moreover, LT-dependent actions on immune cells
could conceivably be relevant for the biology of the arterial
wall [23 –25]. The 5-LO cascade is strongly expressed in
activated macrophages and dendritic cells, LTs activate T
and B cells in vitro, appear to affect skin Langerhans cell
migration to regional lymph nodes in mice [26], and most
cells that participate in immune reactions express LT
receptors. Therefore, LTs may mediate multiple effects on
CV homeostasis. In a clinical study of carotid and coronary
artery, and aorta specimens we observed that 5-LO was
highly expressed in arterial walls of patients afflicted with
clinically significant atherosclerosis [4]. The enzyme
localized to macrophages, dendritic cells, and foam cells
and the number of 5-LO expressing lesion leukocytes
markedly increased during disease progression. These data
support a model of atherogenesis in which 5-LO cascadedependent inflammatory circuits made up by several types
of leukocytes, smooth muscle cells, and endothelial cells
evolve within the blood vessel wall during late stages of
lesion development. According to this model, macrophages
produce LTs which act on neighboring cells to activate their
LT receptors or on the LT receptors of the macrophages
themselves implicating both autocrine and paracrine actions
of LTs (Fig. 1). The 5-LO atherosclerosis hypothesis is
supported by significant expression of all constituents of the
32
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
5-LO pathway and the four LT receptors in human diseased
arteries. One of several target tissues of endogenously
produced LTs in the blood vessel wall may be the
endothelium due to its expression of cysLT2 receptors (see
below). In mice, LTB4 was shown to trigger T cell
recruitment into inflamed tissues [23 – 25]. These data may
indicate that both LTB4 and cysLTs participate in blood
vessel biology. Several questions, however, remain to be
answered: Which cells present in lesions predominantly form
cysLTs and which predominantly form LTB4? Answers to
this question may be relevant to understand the role of LTs in
CV pathophysiology as cysLTs and LTB4 exert very different
activities in blood vessels and each LT receptor may transmit
distinct signaling pathways to modulate the phenotypes of
plaque forming cells (see below). 5-LO+ leukocytes may act
from within the lesions and/or from the adventitial connective tissue in the proximity of cells that express LT
receptors during late stage atherogenesis [4,27]. As 5-LO+
macrophages/dendritic cells/foam cells localize in the
proximity of neoangiogenic blood vessels in areas of
inflammation and in the neighborhood of activated T cells
[4], they may also establish immune response circuits (Fig.
1). Thus, the formation of 5-LO+ cell infiltrates observed in
clinical samples at predilection sites of atherosclerosis, i.e.,
coronary and carotid arteries and the aorta, is compatible
with participation of LTs to endothelial cell dysfunction,
intimal edema, leukocyte infiltration, aberrant contractility,
smooth muscle cell proliferation, and immune reactivity
(Fig. 2).
2. LT receptors may mediate distinct biological responses
in diseased arteries
LTs act through four G protein-coupled 7 membrane
domain-type receptors that appear to be differentially
expressed in normal arterial walls and cells forming
Fig. 2. 5-LO in human atherosclerotic cardiovascular tissues. (A)
Immunohistochemical staining of 5-LO+ cells (red) in type II aorta lesion
according to American Heart Association nomenclature, nuclei are labeled
by hematoxylin (blue). (B) Immunofluorescence staining of 5-LO+ cells
(Cy3, red) next to a small neoangiogenic blood vessel in an intimal type V
lesion of a carotid artery. Endothelial cells were labeled using antisera to
von Willebrand factor (Cy2, green), DNA was stained with DAPI.
atherosclerotic plaques. Two BLT receptors [28,29] and
two cysLT receptors [30,31] have been molecularly characterized and transcripts of all 4 LT receptors have been found
in diseased human arteries [4]. There appears to be a
dichotomy of cysLT receptor expression in blood vessels
and hematopoietic cells: Data indicate preferential expression of cysLT1 receptor in monocyte/macrophages and T
cells and of cysLT2 receptor in endothelial cells [32]. In
addition, though little is known about BLT receptors in the
human normal and diseased arterial wall, BLT1 receptors
were up-regulated in mouse T cell effector cells (cytotoxic
CD8+ effectors, CD8+ central memory cells) and shown to
participate in the recruitment of these T cell effectors to
inflamed tissues in experimental models [23 –25].
2.1. LT-induced gene activation
CysLTs were found to induce sustained Ca++ transients
in human umbilical vein endothelial cells in vitro indicating that LTs are potent endothelial cell activators. We
observed activation of extracellular signal-regulated
kinases 1 and 2 with a maximum at 10 min following
cysLT2 receptor activation (Lötzer and Habenicht, unpublished). To identify the genetic program induced by
cysLT2 receptor-dependent signaling in endothelial cells
and by cysLT1 receptor activation of monocytes/macrophages, microarray expression analyses were carried out
in human umbilical vein endothelial cells and monocytes
(MonoMac6 cells) after addition of LTD4 [27]. These data
revealed that the LTD4-triggered gene activation programs
in the two cell types are very different with only few
overlapping genes: Prominently LTD4-triggered genes in
endothelial cells were macrophage-inflammatory protein 2
(MIP-2), interleukin 8 (IL-8), transcription factor early
growth response 1 and several additional transcription
factors (manuscript in preparation) (Inset in Fig. 1). These
chemokines and their chemokine receptors may exacerbate
CV inflammation by recruiting leukocytes into the arterial
wall. By contrast, in human and mouse monocytic cell
lines, LTD4 did not affect MIP-2 but induced MIP1a, a T
cell chemoattractant [27]. Whereas LTD4’s action in
monocytic cells was inhibitable by the cysLT1 receptor
antagonist, montelukast, its effect on endothelial cells was
not. These data suggested that LTD4 triggers different
responses in an LT receptor and/or target cell-specific
manner. Moreover, a recent study on the effects of LTB4
in cultured monocytes demonstrated that BLT1 receptor
activation mediated induction of a proinflammatory
chemokine, monocyte chemoattractant protein-1 [33].
Taken together, these data indicate that the 5-LO pathway
may act on plaque forming cells and suggest novel
paradigms of CVD inflammation, i.e., the amplification of
leukocyte trafficking and differentiation by the 5-LO
cascade through induction of peptide chemokines. Evidence indicates that both BLT and cysLT receptors may
be involved.
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
2.2. CV Effects in CysLT2-R knockout and transgenic mice
In addition to gene activation, LTs may trigger acute CV
effects independent of transcript regulation. This view is
supported by recent studies using cysLT2 receptor-deficient
[34] and cysLT2 receptor transgenic mice [35]. When the
cysLT2 receptor was deleted in mice the resulting phenotype
showed decreased vascular permeability and attenuated
bleomycin-induced lung fibrosis [34]. The latter finding
may also be relevant to vascular fibroproliferative responses
similar to that observed during atherosclerotic plaque
formation and arterial wall remodeling [36]. In the other
mouse study, the cysLT2 receptor was overexpressed in
endothelial cells. The resulting phenotype also suggested a
role of cysLT2 receptor in blood vessel permeability and
indicated that it participates in blood pressure regulation
[35]. These mouse models should now allow characterization of additional cysLT2 receptor-mediated events in CV
responses to evaluate the potential impact of cysLTs in vivo.
While these recent studies indicate a special role of this LT
receptor subtype in CV homeostasis, the potential interactions of the 5-LO cascade with the arterial wall may be
complex. Further work is required to examine the actions of
LTs on each of the cells that participate in atherogenesis.
33
cell phenotype. That a subpopulation of intima lesion
macrophage/foam cells of human atherosclerotic lesions of
coronary arteries and carotid arteries express dendritic/
Langerhans cell markers further indicates that a subpopulation of lesion macrophages are dendritic cells capable of
presenting antigen to naive T cells thereby promoting
macrophage/dendritic T cell interactions within the arterial
wall [4]. When apoE / mice were fed an atherogenic
diet containing cholesterol, sucrose, and cholic acid,
aneurysms develop in the subdiaphragmatic area of the
abdominal aorta [27,40]. ApoE / /5-LO / mice showed
greatly reduced aortic aneurysm incidence. These data are
consistent with the view that the 5-LO cascade may affect
arterial wall remodeling. We also assayed biomarkers of
inflammation in the circulation and observed that MIP1a
was strongly increased in all hyperlipidemic mice.
Interestingly, 5-LO deletion reduced the extent of MIP1a
up-regulation in a gene dose-dependent fashion. These
results indicated that hyperlipidemia triggers, by unknown
mechanisms, circulating MIP1a levels and that 5-LO
interrupts this up-regulation. These data support hitherto
unrecognized links between 5-LO, hyperlipidemia, and
inflammatory chemokine production in vivo. However,
further work is required to elucidate the underlying
mechanisms of these associations.
3. The 5-LO pathway in hyperlipidemic mice
A locus on mouse chromosome 6, where the 5-LO
gene is located, has been demonstrated to confer
resistance to atherogenesis in the atherosclerosis-resistant
strain CAST/Ei despite hyperlipidemia [37]. It was
hypothesized that 5-LO may represent a pro-atherogenic
susceptibility gene and data in LDL receptor / mice
supported this hypothesis [38]. In addition, pharmacological studies supported a role of LTB4 in the formation of
lipid-rich lesions of the aorta in hyperlipidemic mice [39].
We generated LDL / /5-LO / and apoE / /5-LO /
mice and examined lesion formation, aneurysm formation,
and wire-induced carotid artery responses to injury under
a variety of dietary conditions but failed to observe effects
of 5-LO deletion in lesion formation and intimal
proliferation in response to injury [27] (Nicole John and
A.J.R. Habenicht, unpublished). The reasons for these
differences may relate to different mouse colonies,
immune status, and housing and feeding conditions. When
we examined the abdominal aortas of hyperlipidemic mice
(apoE / ) for 5-LO expression in leukocytes we observed
the presence of two monocyte/macrophage/foam cell
populations: 5-LO+ macrophages accumulate in the
adventitia and 5-LO macrophages accumulate in lipidrich lesions. We noted that intimal macrophages were
CD11c+, whereas adventitial macrophages were CD11cdim.
CD11c is a marker of activated macrophages and of
dendritic cells. These data are consistent with the
possibility that lesion macrophages acquire a dendritic
4. Population genetic studies indicate a role of LTs in
atherogenesis
The Los Angeles Atherosclerosis Study group reported
on patients carrying 5-LO promoter polymorphisms and
related them to CVD [41]. The rationale behind the study
stems from previously described polymorphisms in the 5LO promoter that had been correlated with 5-LO inhibitor
responsiveness in asthma patients and had been presumed to
represent loss of function mutations [42]. Carriers of two
variant alleles exhibited significantly increased atherosclerosis as assessed by sonographic determination of the
intima/media thickness of the common carotid artery.
Moreover, intake of N3 polyunsaturated fatty acids (eicosapentaenoic and docosahexaenoic acid) blunted the effect in
the 5-LO gene variant carriers whereas dietary intake of the
N6 polyunsaturated fatty acids (arachidonic acid and its
precursor linoleic acid) enhanced the effect. The data lend
support to the hypothesis that these fatty acids compete as
substrates for the 5-LO enzyme reaction in vivo and thereby
shift the balance of pro- versus anti-inflammatory 5-LOderived eicosanoids. When taken together, these data
suggest links between the 5-LO pathway and atherogenesis
and between 5-LO and dietary fatty acid intake. However,
we agree with the authors in their cautious interpretation of
these data pointing out several caveats: there was only a
small number of patients carrying the respective variant
alleles making the epidemiological data difficult to interpret;
no biochemical parameters of 5-LO activity were deter-
34
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
mined in these patients but rather, functionality was inferred
from the earlier pharmacological study on asthma patients
[42]: Promoter reporter transfection studies in cultured cells
had yielded functional effects of variant alleles but it
remained uncertain whether any of these variants is
biologically relevant in relation to LT synthesis in vivo
[43 –45]. Thus, functionality of 5-LO promoter genotypes
with respect to LT formation in relevant leukocyte populations has not yet been clearly demonstrated in asthma or
CVD patients.
5. FLAP genotypes correlate with stroke and/or
myocardial infarction
Further support for a link between the 5-LO cascade and
atherogenesis emerged when investigators from deCODE
Genetics reported results of a clinical genetic trial of FLAP
polymorphisms. They mapped a gene that increases risk of
myocardial infarction and stroke to 13q12 –13 containing
the FLAP locus [46]. Since FLAP is believed to be required
for LT biosynthesis probably by acting as an arachidonic
acid binding and transfer protein, FLAP genetic alterations
could result in altered LT formation. In a cohort of 779
patients in Iceland, a particular FLAP gene haplotype
(HapA) was associated with a 2-fold greater risk of
myocardial infarction and stroke while another cohort in
the UK carrying another variant haplotype correlated with
an increased risk of myocardial infarction but not stroke
[46]. Though the authors determined ex vivo LTB 4
formation in neutrophils, LTB4 levels of the haplotype
carriers with myocardial infarction when compared to LTB4
levels of their control myocardial infarction counterparts
were not different. Hence, similar to the Los Angeles study
on 5-LO promoter polymorphisms, a link between FLAP
haplotype and LT formation has not yet been achieved.
However, inherent difficulties of ex vivo LT synthesis
assays that rely on added ionomycin (a rather strong and
unphysiological receptor-independent agonist of LT formation) may have prevented detection of small but functionally
important differences. To establish functionality of the
genotypes, FLAP assays that are sensitive enough to pick
up slight changes in agonist-dependent LT synthesis need to
be established to be performed in clinical studies. Meanwhile, the deCODE Genetics investigators reported on a
Scottish population of 450 patients with ischemic stroke and
710 controls from Aberdeenshire in which genotyping of
seven small nucleotide FLAP polymorphisms was conducted [47]. The Scottish carriers had a significantly greater
stroke risk when compared to the controls. Recently,
Lõhmussaar et al. showed that sequence variants of FLAP
(distinct from the HapA variants reported by deCODE
Genetics) in 639 stroke patients in Southern Germany
showed a weak correlation with stroke whereas the HapA
carriers in the German population did not [48]. When taken
together, these studies are consistent with but clearly not yet
prove connections between genetic alterations of constituents of the 5-LO cascade and CVD. Following this
hypothesis, the deCODE Genetics investigators studied 191
patients with the HapA haplotypes or a newly reported
nucleotide polymorphism in the LTA4 hydrolase gene that
had experienced a myocardial infarction [49]. The study was
designed to assess clinical safety and efficacy of three doses
of the FLAP inhibitor, DG-031, and to determine the effects
of the drug on clinical parameters of CVD risk such as
myeloperoxidase and C-reactive protein [49]. DG-031 was
found to be safe, reduced LTB4 formation in the neutrophil
ex vivo assay by 25%, and also reduced myeloperoxidase
by 12%, whereas C-reactive protein was not affected by the
drug. deCODE Genetics announced initiation of a large
phase III 2 year outcome study of 1500 patients with
previous myocardial infarction to test whether FLAP
inhibition will prevent recurrent heart attacks. If the
upcoming trials to be conducted between 2005 and 2007
confirm the data of the phase IIa trial and indicate beneficial
effects on clinical outcome, conditions for larger drug
development programs to treat arterial inflammation using
anti-LT drugs will be set.
6. The 5-LO and FLAP population studies reveal that
the 5-LO inflammation/atherosclerosis connection may
be clinically significant: what may be the underlying
mechanism?
In the 5-LO promoter polymorphism and FLAP haplotype studies, variant allele carriers were presumed to
represent loss of function [41] and gain of function [46]
phenotypes, respectively, that were assumed to result in a
decrease or increase in LT synthesis in vivo, respectively.
However, carriers of both 5-LO and FLAP haplotypes had
an increased risk to develop carotid artery disease [41] or
myocardial infarction/stroke [46] raising a paradox in the
genetic trials: In fact, these results shed doubt on a close
connection between LT synthesis and CVD. Moreover, the
LT synthesis assays did not reveal significant changes in LT
biosynthesis in ex vivo isolated neutrophils in the FLAP
haplotype carriers (see online Table 7 of reference [46]).
Clearly, if LTs were promoters of CVD inflammation and in
that attribute pro-atherogenic, one would expect a decrease
in CVD in the variant 5-LO allele carriers and an increase in
CVD risk in the HapA carriers in the deCODE Genetics
study. Thus, both studies have fallen short in establishing
alterations of LT synthesis due to genetic alterations. An
alternative explanation, contrary to current belief, is that LTs
may have anti-atherogenic properties in the arterial wall and
a loss of 5-LO function then would result in a decrease in
the defense mechanisms that may normally protect the
arterial wall. A further interpretation of the 5-LO population
studies would be that all variants are associated with gain of
function mutations. These apparent inconsistencies and
loose ends need to be addressed to link genotypes to LT
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
synthesis and LT synthesis to atherosclerosis. As pointed out
above, unless reliable assays can determine the functionality
of 5-LO/FLAP genotypes with respect to LT synthesis in
vivo, little can be said about the molecular basis for the
haplotype/clinical outcome results. Clearly, the data would
be more compelling if assays of LT synthesis that reflect LT
actions in vivo could be associated with the genetic
alterations in the population studies. Any coherent 5-LO
atherosclerosis hypothesis can only emerge after clarification of these important issues.
7. The 5-LO atherosclerosis hypothesis invites further in
depth mechanistic studies into the role of LTs in vascular
wall inflammation
Results of recent human and mouse studies are
consistent with a role of the 5-LO cascade in CVD biology
and arterial wall remodeling. The body of evidence
supporting this hypothesis has increased considerably but
largely relies on the biological activities of high concentrations of added LTs to experimental systems and on the
putative pro-inflammatory roles of LTs in asthma. The
preliminary clinical trials of 5-LO and FLAP gene
variations raise the issue of the underlying molecular
mechanisms as well as the functionality of the genetic
variants of 5-LO cascade constituents. Thus, it will be
important to: (i) examine the relationships between
genotypes and 5-LO and/or FLAP activities in vivo by
developing reliable test systems that allow to determine
subtle changes of LT production in patients afflicted with
CVD to perform prospective clinical trials, a task that may
be difficult to achieve; (ii) test mouse models of CVD and
remodeling in various contexts of injury/inflammation; and
(iii) evaluate impacts of tissue-specific 5-LO pathway
constituents and LT receptor gene deletions in mice; (iv)
identify LT-triggered gene signatures in cultured cells such
as smooth muscle cells, endothelial cells, and hematopoietic cells to define possible in vivo candidate LTregulated genes. Once associations between genetic alterations and functionality will have been established,
pharmacological intervention trials of patients with CVD
will help to determine therapeutic potentials of anti-LT
drugs. Testing of anti-LT drugs in selected patient groups
for drugs, whose safety has been confirmed for the
treatment of chronic lung disease (i.e. in patients that often
are also afflicted with CVD), is possible now including
cysLT1 receptor antagonists and 5-LO inhibitors [50,51].
Additional molecules in the 5-LO cascade deserve attention
as potential drug targets: Inhibitors of LTA4 hydrolase will
selectively limit LTB4 production and inhibitors of LTC4
synthase will reduce the burden of cysLTs. Finally,
development programs for BLT receptor antagonists and
cysLT2 receptor antagonists targeting specific effector sites
of the 5-LO cascade should be invigorated for animal and
subsequent clinical testing. As these approaches are
35
presently leaving the realm of theory, the question
regarding the validity of the 5-LO atherosclerosis hypothesis may be answered in the not too distant future.
Acknowledgements
The manuscript was supported by grants of the Deutsche
Forschungsgemeinschaft (Ha 1083/13-3/13-4/14-1/14-2/125/12-6) and the Interdisziplinäres Zentrum für Klinische
Forschung Jena (TP4.4; TP4.9) and the National Institutes
of Health (HL58464), Canadian Institutes of Health
Research (MOP-67146 and MOP-68930). CDF is holder
of a Canada Research Chair in Molecular, Cellular and
Physiological Medicine. The authors thank Jilly Evans for
comments on the manuscript and Rolf Graebner for Fig. 2.
They also apologize to their colleagues for the omission of
many references due to space restrictions.
References
[1] A.W. Ford-Hutchinson, Leukotriene B4 in inflammation, Crit. Rev.
Immunol. 10 (1990) 1 – 12.
[2] C.D. Funk, Prostaglandins and leukotrienes: advances in eicosanoid
biology, Science 294 (2001) 1871 – 1875.
[3] B. Samuelsson, Leukotrienes: mediators of immediate hypersensitivity
reactions and inflammation, Science 220 (1983) 568 – 575.
[4] R. Spanbroek, R. Grabner, K. Lotzer, M. Hildner, A. Urbach, K.
Ruhling, M.P. Moos, B. Kaiser, T.U. Cohnert, T. Wahlers, A. Zieske, G.
Plenz, H. Robenek, P. Salbach, H. Kuhn, O. Radmark, B. Samuelsson,
A.J. Habenicht, Expanding expression of the 5-lipoxygenase pathway
within the arterial wall during human atherogenesis, Proc. Natl. Acad.
Sci. U. S. A. 100 (2003) 1238 – 1243.
[5] R. Ross, Atherosclerosis—An inflammatory disease, N. Engl. J. Med.
340 (1999) 115 – 126.
[6] J.A. Burke, R. Levi, Z.G. Guo, E.J. Corey, Leukotrienes C4, D4
and E4: effects on human and guinea-pig cardiac preparations in
vitro, J. Pharmacol. Exp. Ther. 221 (1982) 235 – 241.
[7] Y.H. Datta, M. Romano, B.C. Jacobson, D.E. Golan, C.N. Serhan,
B.M. Ewenstein, Peptido-leukotrienes are potent agonists of von
Willebrand factor secretion and P-selectin surface expression in human
umbilical vein endothelial cells, Circulation 92 (1995) 3304 – 3311.
[8] E.B. Friedrich, A.M. Tager, E. Liu, A. Pettersson, C. Owman, L.
Munn, A.D. Luster, R.E. Gerszten, Mechanisms of leukotriene B4triggered monocyte adhesion, Arterioscler. Thromb. Vasc. Biol. 23
(2003) 1761 – 1767.
[9] M. Heimburger, J.E. Palmblad, Effects of leukotriene C4 and D4,
histamine and bradykinin on cytosolic calcium concentrations and
adhesiveness of endothelial cells and neutrophils, Clin. Exp. Immunol.
103 (1996) 454 – 460.
[10] A.J. Huang, J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser,
S.C. Silverstein, Endothelial cell cytosolic free calcium regulates
neutrophil migration across monolayers of endothelial cells, J. Cell
Biol. 120 (1993) 1371 – 1380.
[11] T.M. McIntyre, G.A. Zimmerman, S.M. Prescott, Leukotrienes C4 and
D4 stimulate human endothelial cells to synthesize platelet-activating
factor and bind neutrophils, Proc. Natl. Acad. Sci. U. S. A. 83 (1986)
2204 – 2208.
[12] L. Palmberg, H.E. Claesson, J. Thyberg, Leukotrienes stimulate
initiation of DNA synthesis in cultured arterial smooth muscle cells,
J. Cell Sci. 88 (Pt. 2) (1987) 151 – 159.
36
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
[13] K.E. Pedersen, B.S. Bochner, B.J. Undem, Cysteinyl leukotrienes
induce P-selectin expression in human endothelial cells via a nonCysLT1 receptor-mediated mechanism, J. Pharmacol. Exp. Ther. 281
(1997) 655 – 662.
[14] E. Porreca, C. Di Febbo, A. Di Sciullo, D. Angelucci, M. Nasuti, P.
Vitullo, M. Reale, P. Conti, F. Cuccurullo, A. Poggi, Cysteinyl
leukotriene D4 induced vascular smooth muscle cell proliferation: a
possible role in myointimal hyperplasia, Thromb. Haemost. 76 (1996)
99 – 104.
[15] S. Allen, M. Dashwood, K. Morrison, M. Yacoub, Differential
leukotriene constrictor responses in human atherosclerotic coronary
arteries, Circulation 97 (1998) 2406 – 2413.
[16] S.P. Allen, A.H. Chester, P.J. Piper, A.P. Sampson, E.S. Akl, M.H.
Yacoub, Effects of leukotrienes C4 and D4 on human isolated
saphenous veins, Br. J. Clin. Pharmacol. 34 (1992) 409 – 414.
[17] S.E. Dahlen, J. Bjork, P. Hedqvist, K.E. Arfors, S. Hammarstrom, J.A.
Lindgren, B. Samuelsson, Leukotrienes promote plasma leakage and
leukocyte adhesion in postcapillary venules: in vivo effects with
relevance to the acute inflammatory response, Proc. Natl. Acad. Sci.
U. S. A. 78 (1981) 3887 – 3891.
[18] F. Michelassi, L. Landa, R.D. Hill, E. Lowenstein, W.D. Watkins, A.J.
Petkau, W.M. Zapol, Leukotriene D4: a potent coronary artery
vasoconstrictor associated with impaired ventricular contraction,
Science 217 (1982) 841 – 843.
[19] R.J. Secrest, B.M. Chapnick, Endothelial-dependent relaxation
induced by leukotrienes C4, D4, and E4 in isolated canine arteries,
Circ. Res. 62 (1988) 983 – 991.
[20] G. Smedegard, P. Hedqvist, S.E. Dahlen, B. Revenas, S. Hammarstrom, B. Samuelsson, Leukotriene C4 affects pulmonary and
cardiovascular dynamics in monkey, Nature 295 (1982) 327 – 329.
[21] X.S. Chen, J.R. Sheller, E.N. Johnson, C.D. Funk, Role of
leukotrienes revealed by targeted disruption of the 5-lipoxygenase
gene, Nature 372 (1994) 179 – 182.
[22] J.L. Goulet, R.C. Griffiths, P. Ruiz, R.B. Mannon, P. Flannery, J.L.
Platt, B.H. Koller, T.M. Coffman, Deficiency of 5-lipoxygenase
accelerates renal allograft rejection in mice, J. Immunol. 167 (2001)
6631 – 6636.
[23] K. Goodarzi, M. Goodarzi, A.M. Tager, A.D. Luster, U.H. von
Andrian, Leukotriene B4 and BLT1 control cytotoxic effector T
cell recruitment to inflamed tissues, Nat. Immunol. 4 (2003)
965 – 973.
[24] V.L. Ott, J.C. Cambier, J. Kappler, P. Marrack, B.J. Swanson, Mast
cell-dependent migration of effector CD8+ T cells through production
of leukotriene B4, Nat. Immunol. 4 (2003) 974 – 981.
[25] A.M. Tager, S.K. Bromley, B.D. Medoff, S.A. Islam, S.D. Bercury,
E.B. Friedrich, A.D. Carafone, R.E. Gerszten, A.D. Luster, Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment,
Nat. Immunol. 4 (2003) 982 – 990.
[26] D.F. Robbiani, R.A. Finch, D. Jager, W.A. Muller, A.C. Sartorelli, G.J.
Randolph, The leukotriene C(4) transporter MRP1 regulates CCL19
(MIP-3beta, ELC)-dependent mobilization of dendritic cells to lymph
nodes, Cell 103 (2000) 757 – 768.
[27] L. Zhao, M.P. Moos, R. Grabner, F. Pedrono, J. Fan, B. Kaiser, N.
John, S. Schmidt, R. Spanbroek, K. Lotzer, L. Huang, J. Cui, D.J.
Rader, J.F. Evans, A.J. Habenicht, C.D. Funk, The 5-lipoxygenase
pathway promotes pathogenesis of hyperlipidemia-dependent aortic
aneurysm, Nat. Med. 10 (2004) 966 – 973.
[28] T. Yokomizo, T. Izumi, K. Chang, Y. Takuwa, T. Shimizu, A Gprotein-coupled receptor for leukotriene B4 that mediates chemotaxis,
Nature 387 (1997) 620 – 624.
[29] T. Yokomizo, K. Kato, K. Terawaki, T. Izumi, T. Shimizu, A second
leukotriene B(4) receptor, BLT2. A new therapeutic target in
inflammation and immunological disorders, J. Exp. Med. 192 (2000)
421 – 432.
[30] C.E. Heise, B.F. O’Dowd, D.J. Figueroa, N. Sawyer, T. Nguyen, D.S.
Im, R. Stocco, J.N. Bellefeuille, M. Abramovitz, R. Cheng, D.L.
Williams Jr., Z. Zeng, Q. Liu, L. Ma, M.K. Clements, N. Coulombe, Y.
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
Liu, C.P. Austin, S.R. George, G.P. O’Neill, K.M. Metters, K.R.
Lynch, J.F. Evans, Characterization of the human cysteinyl leukotriene
2 receptor, J. Biol. Chem. 275 (2000) 30531 – 30536.
K.R. Lynch, G.P. O’Neill, Q. Liu, D.S. Im, N. Sawyer, K.M.
Metters, N. Coulombe, M. Abramovitz, D.J. Figueroa, Z. Zeng,
B.M. Connolly, C. Bai, C.P. Austin, A. Chateauneuf, R. Stocco,
G.M. Greig, S. Kargman, S.B. Hooks, E. Hosfield, D.L. Williams
Jr., A.W. Ford-Hutchinson, C.T. Caskey, J.F. Evans, Characterization
of the human cysteinyl leukotriene CysLT1 receptor, Nature 399
(1999) 789 – 793.
K. Lotzer, R. Spanbroek, M. Hildner, A. Urbach, R. Heller, E.
Bretschneider, H. Galczenski, J.F. Evans, A.J. Habenicht, Differential
leukotriene receptor expression and calcium responses in endothelial
cells and macrophages indicate 5-lipoxygenase-dependent circuits of
inflammation and atherogenesis, Arterioscler. Thromb. Vasc. Biol. 23
(2003) e32 – e36.
L. Huang, A. Zhao, F. Wong, J.M. Ayala, M. Struthers, F.
Ujjainwalla, S.D. Wright, M.S. Springer, J. Evans, J. Cui, Leukotriene B4 strongly increases monocyte chemoattractant protein-1 in
human monocytes, Arterioscler. Thromb. Vasc. Biol. 24 (2004)
1783 – 1788.
T.C. Beller, A. Maekawa, D.S. Friend, K.F. Austen, Y. Kanaoka,
Targeted gene disruption reveals the role of the cysteinyl leukotriene
2 receptor in increased vascular permeability and in bleomycininduced pulmonary fibrosis in mice, J. Biol. Chem. 279 (2004)
46129 – 46134.
Y. Hui, Y. Cheng, I. Smalera, W. Jian, L. Goldhahn, G.A. Fitzgerald,
C.D. Funk, Directed vascular expression of human cysteinyl leukotriene 2 receptor modulates endothelial permeability and systemic
blood pressure, Circulation 110 (2004) 3360 – 3366.
M.R. Ward, G. Pasterkamp, A.C. Yeung, C. Borst, Arterial remodeling. Mechanisms and clinical implications, Circulation 102 (2000)
1186 – 1191.
M. Mehrabian, J. Wong, X. Wang, Z. Jiang, W. Shi, A.M. Fogelman,
A.J. Lusis, Genetic locus in mice that blocks development of
atherosclerosis despite extreme hyperlipidemia, Circ. Res. 89 (2001)
125 – 130.
M. Mehrabian, H. Allayee, J. Wong, W. Shi, X.P. Wang, Z.
Shaposhnik, C.D. Funk, A.J. Lusis, Identification of 5-lipoxygenase
as a major gene contributing to atherosclerosis susceptibility in mice,
Circ. Res. 91 (2002) 120 – 126.
R.J. Aiello, P.A. Bourassa, S. Lindsey, W. Weng, A. Freeman, H.J.
Showell, Leukotriene B4 receptor antagonism reduces monocytic
foam cells in mice, Arterioscler. Thromb. Vasc. Biol. 22 (2002)
443 – 449.
A. Daugherty, L.A. Cassis, Mouse models of abdominal aortic
aneurysms, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 429 – 434.
J.H. Dwyer, H. Allayee, K.M. Dwyer, J. Fan, H. Wu, R. Mar, A.J.
Lusis, M. Mehrabian, Arachidonate 5-lipoxygenase promoter genotype, dietary arachidonic acid, and atherosclerosis, N. Engl. J. Med.
350 (2004) 29 – 37.
J.M. Drazen, C.N. Yandava, L. Dube, N. Szczerback, R. Hippensteel, A. Pillari, E. Israel, N. Schork, E.S. Silverman, D.A. Katz, J.
Drajesk, Pharmacogenetic association between ALOX5 promoter
genotype and the response to anti-asthma treatment, Nat. Genet. 22
(1999) 168 – 170.
K.H. In, K. Asano, D. Beier, J. Grobholz, P.W. Finn, E.K.
Silverman, E.S. Silverman, T. Collins, A.R. Fischer, T.P. Keith, K.
Serino, S.W. Kim, G.T. De Sanctis, C. Yandava, A. Pillari, P. Rubin,
J. Kemp, E. Israel, W. Busse, D. Ledford, J.J. Murray, A. Segal, D.
Tinkleman, J.M. Drazen, Naturally occurring mutations in the
human 5-lipoxygenase gene promoter that modify transcription
factor binding and reporter gene transcription, J. Clin. Invest. 99
(1997) 1130 – 1137.
E.S. Silverman, J.M. Drazen, Genetic variations in the 5-lipoxygenase
core promoter. Description and functional implications, Am. J. Respir.
Crit. Care Med. 161 (2000) S77 – S80.
K. Lötzer et al. / Biochimica et Biophysica Acta 1736 (2005) 30 – 37
[45] E.S. Silverman, J. Du, G.T. De Sanctis, O. Radmark, B. Samuelsson,
J.M. Drazen, T. Collins, Egr-1 and Sp1 interact functionally with the
5-lipoxygenase promoter and its naturally occurring mutants, Am. J.
Respir. Cell Mol. Biol. 19 (1998) 316 – 323.
[46] A. Helgadottir, A. Manolescu, G. Thorleifsson, S. Gretarsdottir, H.
Jonsdottir, U. Thorsteinsdottir, N.J. Samani, G. Gudmundsson, S.F.
Grant, G. Thorgeirsson, S. Sveinbjornsdottir, E.M. Valdimarsson, S.E.
Matthiasson, H. Johannsson, O. Gudmundsdottir, M.E. Gurney, J.
Sainz, M. Thorhallsdottir, M. Andresdottir, M.L. Frigge, E.J. Topol,
A. Kong, V. Gudnason, H. Hakonarson, J.R. Gulcher, K. Stefansson,
The gene encoding 5-lipoxygenase activating protein confers risk of
myocardial infarction and stroke, Nat. Genet. 36 (2004) 233 – 239.
[47] A. Helgadottir, S. Gretarsdottir, D. St Clair, A. Manolescu, J. Cheung,
G. Thorleifsson, A. Pasdar, S.F. Grant, L.J. Whalley, H. Hakonarson,
U. Thorsteinsdottir, A. Kong, J. Gulcher, K. Stefansson, M.J.
MacLeod, Association between the gene encoding 5-lipoxygenaseactivating protein and stroke replicated in a Scottish population, Am.
J. Hum. Genet. 76 (2005) 505 – 509.
37
[48] E. Lohmussaar, A. Gschwendtner, J.C. Mueller, T. Org, E. Wichmann,
G. Hamann, T. Meitinger, M. Dichgans, ALOX5AP gene and the
PDE4D gene in a central European population of stroke patients,
Stroke 36 (2005) 731 – 736.
[49] H. Hakonarson, S. Thorvaldsson, A. Helgadottir, D. Gudbjartsson, F.
Zink, M. Andresdottir, A. Manolescu, D.O. Arnar, K. Andersen, A.
Sigurdsson, G. Thorgeirsson, A. Jonsson, U. Agnarsson, H. Bjornsdottir, G. Gottskalksson, A. Einarsson, H. Gudmundsdottir, A.E.
Adalsteinsdottir, K. Gudmundsson, K. Kristjansson, T. Hardarson, A.
Kristinsson, E.J. Topol, J. Gulcher, A. Kong, M. Gurney, G.
Thorgeirsson, K. Stefansson, Effects of a 5-lipoxygenase-activating
protein inhibitor on biomarkers associated with risk of myocardial
infarction: a randomized trial, JAMA 293 (2005) 2245 – 2256.
[50] R. De Caterina, A. Zampolli, From asthma to atherosclerosis-5lipoxygenase, leukotrienes, and inflammation, N. Engl. J. Med. 350
(2004) 4 – 7.
[51] M. Mehrabian, H. Allayee, 5-lipoxygenase and atherosclerosis, Curr.
Opin. Lipidol. 14 (2003) 447 – 457.