Lysophosphatidic acid in atherosclerotic diseases.

Abstract

Lysophosphatidic acid (LPA) is a potent bioactive phospholipid. As many other biological active lipids, LPA is an autacoid: it is formed locally on demand, and it acts locally near its site of synthesis. LPA has a plethora of biological activities on blood cells (platelets, monocytes) and cells of the vessel wall (endothelial cells, smooth muscle cells, macrophages) that are all key players in atherosclerotic and atherothrombotic processes. The specific cellular actions of LPA are determined by its multifaceted molecular structures, the expression of multiple G-protein coupled LPA receptors at the cell surface and their diverse coupling to intracellular signalling pathways. Numerous studies have now shown that LPA has thrombogenic and atherogenic actions. Here, we aim to provide a comprehensive, yet concise, thoughtful and critical review of this exciting research area and to pinpoint potential pharmacological targets for inhibiting thrombogenic and atherogenic activities of LPA. We hope that the review will serve to accelerate knowledge of basic and clinical science, and to foster drug development in the field of LPA and atherosclerotic/atherothrombotic diseases.

Full text

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REVIEWbph_2021
Lysophosphatidic acid in
atherosclerotic diseases
465..482
Andreas Schober1and Wolfgang Siess2
1Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany,
and2Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich,
Germany
Correspondence
Dr Wolfgang Siess, Institute for
Prevention of Cardiovascular
Diseases, University of Munich,
Pettenkoferstr. 9, 80336 Munich,
Germany. E-mail:
wsiess@med.uni-muenchen.de
----------------------------------------------------------------
Keywords
Lysophosphatidic acid; LPA
receptor; endothelial cells;
smooth muscle cells;
macrophage; platelet; neointima;
hyperlipidaemia; chemokine;
atherothrombosis
----------------------------------------------------------------
Received
1 March 2012
Revised
20 April 2012
Accepted
30 April 2012
Lysophosphatidic acid (LPA) is a potent bioactive phospholipid. As many other biological active lipids, LPA is an autacoid: it is
formed locally on demand, and it acts locally near its site of synthesis. LPA has a plethora of biological activities on blood cells
(platelets, monocytes) and cells of the vessel wall (endothelial cells, smooth muscle cells, macrophages) that are all key players
in atherosclerotic and atherothrombotic processes. The specific cellular actions of LPA are determined by its multifaceted
molecular structures, the expression of multiple G-protein coupled LPA receptors at the cell surface and their diverse coupling
to intracellular signalling pathways. Numerous studies have now shown that LPA has thrombogenic and atherogenic actions.
Here, we aim to provide a comprehensive, yet concise, thoughtful and critical review of this exciting research area and to
pinpoint potential pharmacological targets for inhibiting thrombogenic and atherogenic activities of LPA. We hope that the
review will serve to accelerate knowledge of basic and clinical science, and to foster drug development in the field of LPA and
atherosclerotic/atherothrombotic diseases.
Abbreviations
APT1, acyl-protein thioesterase 1; ATX, autotaxin; CCL2, chemokine ligand 2; cPA, cyclic phosphatidic acid; CREB,
cAMP response element-binding; Edg, endothelial differentiation gene; Egr, early growth response gene; ESI, electrospray
ionization; ICAM-1, intracellular adhesion molecule-1; LC, liquid chromatography; LCAT, lecithin-cholesterol
acyltransferase; LDL, low-density lipoprotein; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPL,
lysophospholipids; LPP, lipid phosphate phosphatase; Lp-PLA2, lipoprotein-associated PLA2; LPS, lysophosphatidylserine;
LYPLA-I, lysophospholipase A-I; lysoPLD, lysophospholipase D; MCP-1, monocyte chemotactic protein-1; MLC, myosin
light chain; MM–LDL, minimally modified LDL; mox-LDL, mildly oxidized LDL; MS, mass spectrometry; NVAF,
nonvalvular atrial fibrillation; PA, phosphatidic acid; PAF, platelet-activating factor; PAK, p21-activated kinase; PC,
phosphatidylcholine; PI3K, phosphoinositide 3-kinase; PLA, phospholipase A; ox-LDL, oxidized LDL; SBI, silent brain
infarction; S1P, sphingosine 1-phosphate; SMD, somatomedin domain; SPCs, smooth muscle progenitor cells; sPLA2,
secretory PLA2; SRF, serum response factor; TF, tissue factor; VCAM-1, vascular cell adhesion molecule-1; VSMC, vascular
smooth muscle cells
Introduction
Atherosclerosis is a slowly progressing, multifocal, chronic
arterial disease that is characterized by inflammatory and
regenerative processes, which lead to matrix remodelling and
large lipid deposits. The retention of low-density lipoproteins
(LDL) and activation of endothelial cells initiate atheroscle-
rotic lesion formation in the inner layer (intima) of medium-
and large-sized arteries, such as coronary arteries and cere-
bral arteries, predominantly at predilection sites, where the
laminar blood flow is disturbed (Libby et al., 2011; Weber and
Noels, 2011). In early lesions, monocytes are recruited to the
arterial wall, where they engulf lipids and transform into
foam cells. In the advanced stages of lesion formation, a
fibroatheroma develops in the intima that is characterized by
the extracellular accumulation of lipids and a fibrous cap
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Pharmacology
DOI:10.1111/j.1476-5381.2012.02021.x
www.brjpharmacol.org
British Journal of Pharmacology (2012) 167 465–482465
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society

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consisting of vascular smooth muscle cells (VSMCs) and
extracellular matrix proteins. Although patients with athero-
sclerosis may not develop clinical symptoms, the narrowing
of the arterial lumen may compromise the oxygen supply and
result in ischaemia of the tissues that are supplied by the
affected artery, such as the myocardium or the brain. Further-
more, acute thrombotic occlusion of the arterial lumen can
occur following erosion or rupture of atherosclerotic plaques,
which leads to platelet activation and fibrin formation.
These acute atherothrombotic events are potentially life-
threatening sequelae of atherosclerosis as they may lead to
myocardial infarction or stroke (Fuster et al., 2005; Thim
et al., 2008). Percutaneous interventions, such as coronary
stent implantation, can reopen the occluded or narrowed
arteries and limit ischaemic symptoms, and are often life-
saving. However,suchinterventions
re-narrowing of the target vessel (restenosis) due to the accu-
mulation of SMCs in the neointima.
Lysophosphatidic acid (LPA) is a phospholipid that medi-
ates a plethora of activities in blood cells and cells of the
vessel wall (Tigyi, 2001; Siess, 2002; Siess and Tigyi, 2004;
Smyth et al., 2008). Similar to other lipid mediators such as
prostaglandins and leukotrienes, LPA acts as an autacoid.
After local formation in response to danger signals such as
those initiated by vascular injury or inflammation LPA
rapidly activates cells in the immediate vicinity (Tokumura,
1995). The action of LPA is mediated by its binding to and
activation of surface GPCRs, including the Edg family of LPA
receptors LPA1–3, LPA4 (GPR23), LPA5 (GPR92), and LPA6
(P2Y5), the latter three belonging to the purinoceptor cluster
(Choi et al., 2010; Chun et al., 2010; Tigyi, 2010). Currently
putative LPA GPCRs are GPR87, P2Y10 and GPR35; hence,
there are in total nine LPA receptors (Choi et al., 2010; Chun
et al., 2010; Tigyi, 2010). In addition to GPCRs, some of the
effects of LPA appear to be mediated by the activation of the
nuclear receptor PPARg, specifically in macrophages and
VSMCs (McIntyre et al., 2003; Zhang et al., 2004).
OurreviewisfocusedontheroleofLPAanditsreceptorsin
atherosclerosis and cardiovascular diseases. Since the seminal
observations that LPA is generated during mild oxidation of
LDL and accumulates in the lipid core of human atheroscle-
rotic plaques linking, for the first time, LPA to atherosclerosis
(Siesset al.,1999),theareahasevolveddramatically.Weaimto
cover this research field in the present review. General aspects
of LPA receptor functions and their coupling to intracellular
signalling pathways (Choi et al., 2010; Tigyi, 2010; Yanagida
and Ishii, 2011), of intra- and extracellular LPA formation and
metabolism (Nakanaga et al., 2010; Samadi et al., 2011; van
Meeteren and Moolenaar, 2007), and of the role of LPA in
cardiovascular physiology (Smyth et al., 2008) have been
excellently reviewed previously.
can resultina
LPA generation in vascular diseases
Although the absolute concentration of circulating LPA varies
considerably in different assays (Smyth et al., 2008), it is clear
that LPA is detectable in plasma in the low mM range. The
generation of circulating LPA essentially requires the action of
autotaxin (ATX), a secreted lysophospholipase D, which
removes the polar head group from lysophospholipids (LPL),
such as lysophosphatidylcholine (LPC) (Umezu-Goto et al.,
2002; Tokumura et al., 2002c; Nakanaga et al., 2010). Accord-
ingly, plasma LPA levels of healthy subjects strongly correlate
with the serum ATX activity (Hosogaya et al., 2008) and are
reduced by 50% in mice heterozygous for ATX (Tanaka et al.,
2006; van Meeteren et al., 2006). Moreover, depletion of ATX
completely prevents LPA production in serum (Tanaka et al.,
2006; Tsuda et al., 2006), and ATX overexpression in trans-
genic mice increases LPA plasma levels (Pamuklar et al., 2009).
Inhibition studies demonstrated that continual ATX activity
is required to maintain a steady state concentration of LPA in
the circulation (Albers et al., 2010; Gierse et al., 2010; Gupte
et al., 2011). A boronic acid-based inhibitor in mice resulted in
a rapid decline of LPA within minutes and the orally available
ATX antagonist PF-8380 dose-dependently diminished the
plasma LPA concentration by maximally 70% within 3 h
(Albers et al., 2010; Gierse et al., 2010).
Adipocyte-derived ATX generates almost half of the LPA
in plasma (Dusaulcy et al., 2011). In obesity, insulin resist-
ance in adipocytes has been linked to increased expression of
ATX, implicating a potential role of LPA in the sequelae of the
metabolic syndrome (Boucher et al., 2005). Furthermore,
hyperlipidaemia enhances the activity of circulating ATX in
rabbits, which leads to enhanced generation of LPA from LPC
in serum, and increases plasma LPA and adipose tissue expres-
sion of ATX in mice fed a high fat diet (Tokumura et al.,
2002a; Dusaulcy et al., 2011).
Elevated circulating LPA levels may have pro-atherogenic
effects, because systemic treatment with unsaturated LPAs has
been shown to enhance atherosclerosis (Zhou et al., 2011). In
humans, plasma LPA levels correlate with LPC, ATX and LDL
levels, indicating that hyperlipidaemia may contribute to
the generation of LPA in the circulation (Dohi et al., 2012).
Moreover, circulating LPC is increased by hyperlipidaemia
(Portman et al., 1970; McConnell and Hoefner, 2006; Mat-
sumoto et al., 2007; Schmitz and Ruebsaamen, 2010). Plasma
LPC, which is mainly bound to albumin is generated by
endothelial lipase and by lecithin-cholesterol acyltransferase
(LCAT) secreted from the liver (Wiesner et al., 2009; Schmitz
and Ruebsaamen, 2010). Prolonged (10–50 h) incubation of
plasma at 37°C leads to a large increase in LPA from 0.5 to
15 mM that is due to LPC generation by LCAT and not by
lipoprotein-associated (Lp-) PLA2activity (Aoki et al., 2002).
Progression of atherosclerosis is associated with condi-
tions of chronic inflammation and oxidant stress. Under
these conditions, circulating LPC can be generated by group
IIA secretory PLA2(sPLA2), which is an acute-phase reactant,
and by Lp-PLA2 (identical to platelet-activating factor (PAF)
acetylhydrolase) (Stafforini, 2009; Rosenson, 2010). sPLA2
acts on lipoproteins and microvesicles (Fourcade et al., 1995),
whereas Lp-PLA2specifically catalyses the removal of the acyl
group at the sn-2 position of oxidatively truncated phospholi-
pids present in oxidized LDL (ox-LDL) (Steinbrecher et al.,
1984). LDL particles oxidized to different degrees have been
detected at increased levels in the circulation of patients
with acute coronary syndromes (Holvoet et al., 1998).
Furthermore, electronegative LDL particles, which are pro-
inflammatory by releasing chemokines from endothelial
cells, are increased and physicochemically heterogeneous in
familial hypercholesterolaemic patients (Sanchez-Quesada
et al., 2002; 2003). Moreover, circulating levels and enzymatic
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activity of Lp-PLA2 and sPLA2 have been found to predict
cardiovascular events (Packard et al., 2000; Koenig and Khu-
seyinova, 2009; Thompson et al., 2010). Lp-PLA2is primarily
bound to electronegative LDL in the circulation, and the
levels of Lp-PLA2positively correlate with the concentration
of LDL (Packard et al., 2000; Benitez et al., 2003; Albert et al.,
2005). Of note, the Lp-PLA2inhibitor darapladib, which has
currently been tested in two large cardiovascular outcome
trials, limits atherosclerosis in mice (Charo and Taub, 2011;
Wang et al., 2011). The sPLA2 inhibitor varespladib reduces
atherosclerosis in mice and a phase III cardiovascular
outcome trial on the effects of varespladib is ongoing (Charo
and Taub, 2011).
Although it remains to be determined whether pro-
atherogenic effects of Lp-PLA2 and sPLA2 are related to
increased LPA production, a very recent study showing a
correlation between levels of Lp-PLA2, LPCs, LPA and pro-
inflammatory cytokines in human plaques supports such a
link (Goncalves et al., 2012). Therefore, it can also be assumed
that LPC levels elevated in hyperlipidaemia may lead to
increased ATX-dependent LPA generation in the circulation.
An interesting question in this regard is whether mice defi-
cient in sPLA2 or Lp-PLA2 show decreased cellular LPA pro-
duction or decreased LPA plasma levels. However, apart from
one study that shows normal LPA generation of activated
washed platelet suspensions prepared from mouse strains
that are deficient in sPLA2 expression (such as the C57BL/6
and C3H strains) (le Balle et al., 1999), nothing is known
about this topic.
LPA accumulates in human and mouse atherosclerotic
lesions (Siess et al., 1999; Bot et al., 2010). LPA in atheroscle-
rotic lesions is most likely to be derived from ATX-mediated
hydrolysis of LPC. Although the expression of ATX in athero-
sclerotic lesions has not been studied, it has been shown that
ATX can be secreted from arterial endothelial cells and that it
is up-regulated in the arterial wall following vascular injury
(Panchatcharam et al., 2008; Zhou et al., 2011). Structural
analysis has suggested that ATX is capable of directing the
LPA produced to the cognate GPCRs via binding of its
somatomedin domains (SMD) to b3-integrins, thus contrib-
uting to localized LPA formation (Fulkerson et al., 2011;
Hausmann et al., 2011; Nishimasu et al., 2011). However, it
remains to be determined whether local LPA production and
delivery by ATX via binding to b3-integrins, which are highly
expressed in various cell types of atherosclerotic lesions, con-
tributes to LPA formation in the lesion (Hoshiga et al., 1995).
In atherosclerotic lesions, the ATX substrate LPC is gen-
erated mainly by LDL oxidation. Polyunsaturated fatty acids
in the sn-2 position of phosphatidylcholine (PC) in the outer
layer of the LDL particle undergo oxidative fragmentation to
oxidized short-chain fatty acids (Siess, 2006). The oxidized PC
molecules are then specifically hydrolyzed by Lp-PLA2, which
produces LPC (Steinbrecher et al., 1984). LPC in the vessel
wall accumulates during hyperlipidaemia, and LPC accumu-
lation in a mouse atherosclerotic model precedes the progres-
sive accumulation of LPA in atherosclerotic tissue (Portman
et al., 1970; Bot et al., 2010).
In addition, LPA in atherosclerotic lesions might derive
indirectly from microparticles or microvesicles that are shed
from activated and apoptotic cells (e.g. macrophages and
VSMCs) and accumulate in inflamed atherosclerotic lesions.
Membrane phospholipids of microvesicles, but not intact
cells have been reported to be accessible to hydrolysis by
sPLA2, and the concentration of lysophospholipids was found
to be increased in microvesicles isolated from inflammatory
fluids (Fourcade et al., 1995). Thus, this enzyme could also
generate lysophospholipids from microparticles for ATX-
dependent LPA formation in inflamed atherosclerotic lesions,
although the role of microparticles in the production of
lesional LPA remains to be determined.
Previous reports have described increased formation of
LPA, preferentially of the alkyl-LPA species, during mild oxi-
dation of LDL (Siess et al., 1999; Zhang et al., 2004), suggest-
ing that a pathway of LPA formation during LDL oxidation
may exist, which may be ATX-independent. However, the
possibility that ATX, similar to other plasma proteins, might
also be associated with LDL (Hoofnagle and Heinecke, 2009)
cannot be excluded, although direct evidence is lacking.
Platelet activation importantly contributes to LPA forma-
tion in blood. Platelet depletion reduces the serum LPA levels
by 50% in rats (Aoki et al., 2002). During blood clotting, the
LPA concentration rises from low micromolar plasma concen-
trations (0.1–1 mM) up to 10 mM in serum predominantly by
an increase of acyl-LPA species containing polyunsaturated
fatty acids (mainly 18:2 and 20:4 fatty acids) (Siess, 2002;
Tigyi, 2010). Activation of isolated platelets induces the for-
mation of intracellular LPA via a pathway that involves the
PLC-catalysed hydrolysis of phosphoinositides leading to the
formation of diacylglycerol that is rapidly phosphorylated by
diglyceride kinase to phosphatidic acid (PA) (Siess, 2002).
Subsequently, degradation by platelet PLA2and PLA1enzymes
specific for PA leads to the formation of 1-acyl-LPA and
2-acyl-LPA, respectively (Billah et al., 1981; Gaits et al., 1997).
IntracellularlyproducedLPA
the extracellular medium (Watson et al., 1985). LPA in the
medium of activated platelets can only be detected in the
presence of albumin (Eichholtz et al., 1993). Recent studies
indicate that the small amounts of extracellular LPA that can
be detected upon activation of isolated platelets are due to
the activity of platelet-bound ATX (Pamuklar et al., 2009;
Fulkerson et al., 2011). Thus, it seems likely that extracellular
LPA detected upon activation of washed platelets is formed by
platelet-bound ATX from LPC, and albumin by binding
platelet-released LPC plays an essential role in this process.
In contrast to activation of platelets in buffer, platelet
activation in the presence of plasma leads to a large increase
in extracellular LPA. This occurs by a multistep process
involving the generation of LPL via intracellular PLAs and
secreted PLA1 and/or PLA2 enzymes (Aoki et al., 2002; Sano
et al., 2002). Very recent studies demonstrating the binding of
ATX to b3-integrins of activated platelets (Fulkerson et al.,
2011) and the identification of a new PLA enzyme secreted
from platelets (Bolen et al., 2011) unravel how platelet acti-
vation may lead to the large increase in certain molecular LPA
species (18:2-acyl-LPA and 20:4 acyl-LPA) in serum. The plate-
let PLA enzyme that may feed plasma ATX with LPLs upon
platelet activation is acyl-protein thioesterase 1 (APT1),
also known as lysophospholipase A-I (LYPLA-I) (Bolen
et al., 2011). APT1 is released from activated platelets, acts
like PLA1 and generates a pool of sn-2-esterified lysophos-
pholipids containing mainly C18:2 and C20:4 (Bolen et al.,
2011).Thethermodynamically
isnot released into
unstable
sn-2-esterified
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lysophospholipids undergo acyl migration resulting in sn-
1-esterified lysophospholipids, which are the preferred sub-
strates of ATX (Bolen et al., 2011). Subsequently, ATX bound
to activated b3-integrins on the activated platelet membrane
via its two SMD produces LPA species containing mainly 18:2
and 20:4 fatty acids (Bolen et al., 2011; Fulkerson et al., 2011).
This platelet-dependent LPA production may be crucial for
vascular repair, for example, after erosion of atherosclerotic
plaques or after stent implantation, where activated platelets
rapidly adhere to the denuded vascular surface.
Extracellular LPA is degraded by the ecto-activities of lipid
phosphate phosphatases (LPPs) (Morris et al., 2009; Samadi
et al., 2011). LPPs hydrolyze a large variety of bioactive lipid
phosphates and pyrophosphates; their active site resides on
the outer surface of plasma membranes. Three different mam-
malian LPP isoforms are known. Whereas i.v. injected LPA has
a half-life of only 3 min in mice, an extended half-life of
circulating LPA up to 12 min and increased levels of plasma
LPA have been found in LPP1-deficient mice (Tomsig et al.,
2009). It is therefore likely that a balance between ATX and
LPP activities controls the concentration of extracellular LPA
and thus LPA receptor activation in a dynamic manner
(Samadi et al., 2011).
In addition to LPA, cyclic phosphatidic acid (1-acyl-sn-
glycerol-2,3-cyclic phosphate; cPA) probably formed by ATX,
has been identified in human serum (Kobayashi et al., 1999;
Tsuda et al., 2006; Fujiwara, 2008; Shan et al., 2008b). cPA is
an LPA analogue in which the sn-2 hydroxyl group has
formed a ring structure with the sn-3 phosphate. Although
the function of extracellular cPA on vascular cells and blood
cells is largely unknown (Fujiwara, 2008; Tigyi, 2010), intra-
cellular cPA, formed by PLD activation, functions as inhibitor
of PPARg and might thereby modify atherosclerotic processes
(Tsukahara et al., 2010).
Measurement of plasma LPA and
circulating LPA levels in cardio- and
cerebrovascular clinical studies
The two primary methods that are used to measure LPA in
plasma are liquid chromatography-electrospray ionization
tandem MS (LC-ESI-tandem MS) and enzymatic assays. Two
enzymatic methods have been described: (i) a radioenzymatic
assay uses recombinant LPA acid acyltransferase and a radio-
active fatty acid to measure the formation of radiolabeled
PA (Saulnier-Blache et al., 2000); and (b) an enzymatic
cyclingassayuseslysophospholipase,
glycerol 3-phosphate, followed by enzymatic cycling using
glycerol 3-phosphate oxidase and glycerol 3-phosphate
dehydrogenase. The amplified concentrations of hydrogen
peroxide are then measured colorimetrically (Kishimoto
et al., 2003). Both of these methods yielded LPA plasma levels
(0.07–0.1 mM) that were 10-fold lower than the levels meas-
ured by the LC/MS/MS method (0.7–1 mM) (Baker et al., 2001;
Liebisch and Scherer, 2012).
The determination of plasma LPA is not trivial, and the
conditions of sample preparation are important for accurate
measurement. The majority of LPA in the circulation is bound
to albumin (Tigyi et al., 1991; Tigyi and Miledi, 1992), and
whichgenerates
acidic extraction methods are required. The addition of
strong acids may lead to conversion of plasma LPC to LPA in
plasma samples, leading to an artificial increase in LPA (Shan
et al., 2008a; Liebisch and Scherer, 2012). This is important
because plasma LPC levels are approximately 100–1000 times
higher than that of LPA. To circumvent this problem, the
butanolic extraction procedure (pH 4) described by Baker
et al. is often the method of choice for LPA extraction (Baker
et al., 2001; Liebisch and Scherer, 2012). Furthermore, direct
mass spectrometric analysis of crude lipid extracts should
be avoided when determining the levels of plasma LPA.
In-source fragmentation of LPC and lysophosphatidylserine
(LPS) to LPA has been reported after direct flow injection
during plasma LPA analysis (Zhao and Xu, 2009; Liebisch and
Scherer, 2012). LPC can lose its choline group at the ion
source before the parent ions are detected, giving rise to
signals that are indistinguishable from endogenous LPA.
Activated platelets also contribute significantly to LPA
generation in the blood, which is an additional factor that
makes it difficult to accurately determine the circulating LPA
levels. Platelets are activated easily after venipuncture, and it
is impossible to immediately inhibit platelet activation before
the blood comes into contact with the anticoagulant and
additional platelet inhibitors that are perhaps present in the
blood collection cuvette. Similar problems have previously
been encountered when determining the concentration of
circulating thromboxane and other substances that are
released from activated platelets (FitzGerald et al., 1987).
However, platelet activation during the subsequent handling
of the blood such as during the centrifugation of blood to
obtain plasma can be controlled. The lowest plasma concen-
trations of LPA (approximately 0.1 mM, measured by the
enzymatic cycling assay) (Kishimoto et al., 2003) and LPC
(approximately 190 mM) were found when blood was drawn
into 7.5 mM EDTA plus a mixture of 10% (v v-1) citrate,
theophylline, adenosine and dipyridamole (Nakamura et al.,
2007). The latter three substances are platelet inhibitors. In
blood samples that were collected in this manner, the LPA
concentration was significantly higher in women (0.1 mM)
than in men (0.077 mM), and a positive correlation between
the plasma LPA concentration and serum lysophospholipase
D (lysoPLD) activity was found. However, the LPA concentra-
tion could be correlated with the plasma LPC concentration
only in men (Hosogaya et al., 2008).
In most of the clinical studies that report LPA plasma
levels, no precautions have been taken to control for the
artificial contribution of LPA production from activated plate-
lets in vitro during blood handling and processing. For future
studies, the inclusion of a pharmacological ATX inhibitor
would be ideal to circumvent this problem. After i.v. injection
of a boronic acid-based inhibitor of ATX into mice, a rapid
decrease in the total plasma LPA concentration was observed;
this finding was interpreted to indicate that there is a
dynamic turnover of LPA in the circulation (Albers et al.,
2010). Another explanation for these results could be that the
injected ATX inhibitor attenuated the in vitro formation of
LPA during blood handling.
In a recent cross-sectional study of consecutive patients, it
was reported that patients with acute coronary syndrome
have significantly increased plasma LPA levels (0.54 mM)
compared with patients with stable angina pectoris (0.36 mM)
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or angiographically normal coronary arteries (0.41 mM) (Dohi
et al., 2012). In this study, LPA was measured enzymatically
as previously described (Kishimoto et al., 2003); however,
special precautions to inhibit LPA formation during blood
handling in vitro (Nakamura et al., 2007) were not taken. The
blood was collected into EDTA-containing tubes (Dohi et al.,
2012), and the LPA plasma levels of the patients with angi-
ographicallynormal coronary
0.4 mM) were approximately four times higher than the LPA
plasma levels that have been reported previously in healthy
people. The interpretation of this clinical study is therefore
difficult as patients with unstable angina pectoris might
exhibit higher LPA plasma levels because their platelets are
more easily activated in vitro.
Another clinical study investigated the influence of ace-
tylsalicylate on plasma LPA levels in patients with ischaemic
cerebral vascular diseases (Li et al., 2008). Elevated LPA levels
were found in patients with ischaemic cerebrovascular
disease (3.11 mM) compared with healthy controls (1.77 mM).
Daily administration of aspirin (100 mg) for 1 month signifi-
cantly lowered the LPA levels in the patients from 4.06 to
2.41 mM. The authors concluded that their findings support a
close association between increased plasma LPA levels and
platelet activation. Again, it is difficult to interpret whether
the effect of aspirin on the plasma LPA levels occurred in vivo
or in vitro.
A separate clinical study investigated, whether there is a
relationship between LPA levels and the prevalence of silent
brain infarction (SBI) in patients with non-valvular atrial
fibrillation (NVAF) (Li et al., 2010). The plasma LPA levels in
the NVAF patients with SBI were significantly higher than
those in the control patients (P < 0.01) or the NVAF patients
without SBI. The authors suggested that LPA might be a novel
marker for estimating the status of platelet activation and the
risk for SBI onset in NVAF patients.
The question when interpreting all of these studies is
whether the plasma LPA that is measured truly reflects the
circulating LPA concentration or whether it was formed in
vitro from platelets that were activated during blood han-
dling. In future studies, the addition of a pharmacological
ATX inhibitor to the blood collection tube is recommended
to minimize LPA formation during blood handling. Moreo-
ver, the measurement of total plasma LPA levels may not be
sufficient to predict the cardiovascular risk, because the
thrombogenicity and atherogenicity of LPA crucially depends
on the type of bondage of the fatty acyl chain to the glycerol
backbone (ester or ether) and the saturation of the fatty acid;
alkyl-LPA species are more potent platelet activators than the
corresponding acyl-LPA species (Simon et al., 1982; Tokumura
et al., 2002d; Rother et al., 2003), and only unsaturated acyl-
LPA species are atherogenic (Yoshida et al., 2003; Zhang et al.,
2004; Zhou et al., 2011). In line with these findings, elevated
levels of LPA during pregnancy, which is not associated with
increased cardiovascular risk, is due to a rise predominantly
of saturated LPA, such as LPA16:0 (Tokumura et al., 2002b).
Furthermore, increased circulating LPA has been found in
patients with chronic hepatitis C and liver fibrosis and in
experimentally induced liver
2007a,b). In contrast to hepatitis C infection, which is asso-
ciated with increased cardiovascular risk, liver cirrhosis does
not lead to accelerated atherosclerosis (Petta et al., 2011;
arteries (approximately
fibrosis (Watanabe
et al.,
Purnak et al., 2011; Adinolfi et al., 2012). Unfortunately, it is
currently unknown which LPA species are elevated in the
different forms of liver diseases.
Further studies on the circulating levels of the various LPA
species may help to clarify the role of elevated circulating LPA
levels in atherogenesis. These could also give a hint for the
origin of LPA, because circulating LPA might derive from
activated platelets (which produce mainly 18:2-acyl-LPA and
20:4 acyl-LPA), microparticles originating from damaged or
apoptotic cells (their LPA species are unknown) or other cell
types (adipocytes). However, in general, the relevance of
dosing LPA in plasma could be questioned, because LPA is
mainly generated locally in the circulation and acts locally.
Effects of LPA on blood and
vascular cells
LPA has multiple effects on blood cells and cells of the vessel
wall (Siess, 2002; Smyth et al., 2008). In platelets, it induces
directly shape change, and it stimulates platelet aggregation
and secretion only in synergy with other platelet stimuli
(Rother et al., 2003; Haseruck et al., 2004). In human mono-
cytes, LPA increases cytosolic Ca2+(Fueller et al., 2003), and in
macrophages, it stimulates cell survival and ox-LDL uptake
(Koh et al., 1998; McIntyre et al., 2003). In endothelial cells,
LPA stimulates cell migration (Panetti et al., 2000; 2004;
Ptaszynska et al., 2010), chemokine secretion (Lin et al., 2006;
Zhou et al., 2011), adhesion molecule expression (Rizza et al.,
1999; Zhou et al., 2011), actin stress fibre formation and cell
contraction (Siess et al., 1999; Hirakawa et al., 2004). In con-
fluent endothelial cells in vitro or in endothelium in vivo, LPA
can either increase (van Nieuw Amerongen et al., 2000; Hirase
et al., 2001; Sarker et al., 2010) or decrease (Alexander et al.,
1998; Minnear et al., 2001) endothelial permeability. LPA also
co-operates with VEGF to stimulate angiogenesis (Tanaka
et al., 2006; van Meeteren et al., 2006; Ptaszynska et al., 2010).
In VSMCs, LPA stimulates cell contraction, leading to
increased vascular tone, cell migration, and proliferation [for
ref. see (Siess, 2002; Smyth et al., 2008) ].
The specific cellular effects of LPA depend on the species
of the LPA molecule, and the origin and the vascular envi-
ronment of the target cells. There are multiple molecular
species of LPA that have been described in biological fluids
and that are present in plasma and the lipid-rich core of
atherosclerotic plaques (Baker et al., 2001; Rother et al.,
2003). The fatty acid is mainly attached at the sn-1 position of
the glycerol backbone via an ester (1-acyl-LPA) or ether bond
(1-alkyl-LPA, 1-alkenyl LPA). The fatty acids, which are of
predominantly 16-, 18- and 20-carbon lengths, can be satu-
rated, mono- or poly-unsaturated. Additionally, 2-acyl regio-
isomers of LPA have been detected; however, sn-2-esterified
lysophospholipids are unstable and undergo acyl migration
to yield sn-1-esterified lysophospholipids (Bolen et al., 2011).
The types of LPA molecules that are present determine,
qualitatively or quantitatively, the biological response of the
cell. For example, acyl-LPA that contain unsaturated but not
saturated fatty acid causes the phenotypic dedifferentiation
of cultured VSMCs and elicits neointima formation in a non-
injury model of the rat carotid artery (Hayashi et al., 2001;
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Yoshida et al., 2003; Zhang et al., 2004; Subramanian et al.,
2010). Furthermore, LPA (20:4), but not LPA (18:0), triggers
the adhesion of monocytes to the vessel wall and enhances
the progression of atherosclerosis (Zhou et al., 2011). In plate-
lets, alkyl-LPA (16:0) and acyl-LPA (20:4) are approximately
20- and 7-fold more potent than acyl-LPA (16:0), respectively
(Tokumura et al., 2002d; Rother et al., 2003; Haseruck et al.,
2004). In cells that express individual recombinant LPA recep-
tors, most of the receptors show an agonist preference for
mono- or poly-unsaturated acyl-LPA over saturated acyl-LPA
(Tigyi, 2010). However, whether endogenous LPA receptors,
which are expressed at much lower levels in mammalian
cells, behave similarly is not known.
The type of LPA response can also vary between species.
For example, human and cat platelets, but not rodent (rat and
mouse) platelets, are activated by LPA (Schumacher et al.,
1979; Tokumura et al., 1981). Interestingly, mouse platelets
are inhibited by LPA (Pamuklar et al., 2009). Additionally, the
type of vascular bed is important in considering the effect of
LPA on endothelial cells because the endothelium shows a
pronounced heterogeneity. There are varied biomechanical
and biochemical inputs along the vasculature that alter the
properties of the endothelium, and therefore its response to
the same pathophysiological stimuli will be different (Aird,
2005). Thus, in vivo studies on the action of LPA on endothe-
lial cells will be particularly important as these allow the
study of endothelial cells in healthy and atherosclerotic arte-
rial vessels in situ (Zhou et al., 2011). In contrast, in vitro
culture of endothelial cells cannot preserve their vascular
bed-specific phenotype, and cell culture also leads to a loss of
glycocalyx on the cell surface, which is an important
endothelial interface with circulating blood (Chappell et al.,
2009).
An important consideration when investigating the activ-
ity of circulating LPA is that plasma albumin might limit the
activity of LPA. Albumin binds LPA with a stoichiometry of
3 mol of LPA to 1 mol of albumin (Tigyi and Miledi, 1992;
Thumser et al., 1994), and albumin dose-dependently inhib-
its LPA-induced platelet shape change and aggregation (Toku-
mura et al., 2002d; Haseruck et al., 2004; Khandoga et al.,
2008). This finding explains the observation that approxi-
mately 1000-fold higher LPA concentrations (0.5–20 mM) are
required to stimulate platelets in plasma and blood, as com-
pared with the stimulation of washed platelet suspensions. It
is therefore unlikely that the low LPA concentrations found
in plasma (total LPA: 0.1–1 mM) affect circulating platelets,
and these low levels may also not affect other blood cells or
endothelial cells as well.
For many of the effects of LPA on blood cells and cells of
the vessel wall, the LPA receptors that are responsible have
not been identified. The availability of mice that are deficient
for individual LPA receptors (Choi et al., 2010) has advanced
our knowledge of the involvement of specific LPA receptors
in atherosclerotic processes (Panchatcharam et al., 2008).
Often, in vitro studies of LPA receptor expression in individual
cells have to rely on quantitative PCR analysis of mRNA
transcripts because the possibly low endogenous expression
of LPA receptor proteins is difficult to determine by immu-
noblot with specific LPA-receptor antibodies. Furthermore,
the fact that one cell type often expresses many different LPA
receptors, and that a single LPA receptor can couple to differ-
ent G-proteins in the same cell (Choi et al., 2010), thereby
stimulating a complex LPA signalling network, makes it dif-
ficult to dissect the function of individual LPA receptors.
However, studies using the siRNA technology to down-
regulate individual LPA receptors in vascular cells, blood cells
or isolated arteries are helpful to assign a specific LPA receptor
to its cellular or vascular effect (Subramanian et al., 2010;
Khandoga et al., 2011; Zhou et al., 2011).
LPA in acute atherothrombosis
Myocardial infarction and ischaemic stroke are leading causes
of morbidity and mortality in humans. The trigger for
approximately 70% of myocardial infarctions is plaque
rupture, and the erosion of vulnerable atherosclerotic plaques
accounts for the other 30%. These events lead to the exposure
of thrombogenic plaque material to circulating blood
(Fernandez-Ortiz et al., 1994; van Zanten et al., 1994; Toschi
et al., 1997; Kolodgie et al., 2004). Subsequent platelet activa-
tion and fibrin formation can lead to the development of an
occluding thrombus with possibly fatal consequences for the
patient. LPA may play different roles in thrombus formation
after erosion and plaque rupture. After plaque erosion, the
main thrombogenic stimulus is possibly subendothelial
versican-hyaluronan matrix (Kolodgie et al., 2004), whereas
after plaque rupture, collagen of the disrupted cap and pos-
sibly LPA of the plaque core are the platelet stimuli (van
Zanten et al., 1994; Siess et al., 1999; Corti and Badimon,
2002; Rother et al., 2003; Penz et al., 2005; Nakanaga et al.,
2010). The lipid core contains alkyl-LPA and acyl-LPA species
with high platelet-activating potency (Rother et al., 2003)
(Figure 1). On the other hand, LPA locally formed by ATX
bound to activated platelets that cover eroded plaques might,
in concert with other platelet-derived mediators such as
sphingosine 1-phosphate (S1P) and VEGF, activate neigh-
bouring endothelial cells to migrate and proliferate, and
thereby help in healing the endothelial defect (Panetti et al.,
2000; 2004; Ptaszynska et al., 2010).
LPA that is produced by activated platelets after plaque
erosion or rupture may play a role as a positive feedback
mediator of platelet activation. However, in a previous study,
no inhibitory effects of LPA receptor antagonists were found
on aggregation of washed platelets stimulated by collagen or
thrombin, indicating that the small amounts of LPA that were
generated by stimulated, washed platelets do not mediate or
support stimulus-induced platelet aggregation (Haseruck
et al., 2004). Whether LPA plays a role as a positive feedback
mediator of platelet activation in stimulated blood where
ATX-dependent LPA formation rises drastically is not known.
Thrombin activation of isolated platelets leads to the acti-
vation of the integrin aIIbb3 and binding of extracellular
ATX to the b3 integrin through its tandem somatomedin B
(SMB) domains (Fulkerson et al., 2011; Hausmann et al.,
2011). Echistatin, an arginine–glycine–aspartic acid (RGD)-
containing peptide, and a monoclonal antibody against
integrin aIIbb3 (10E5) reduced platelet adhesion to ATX and
inhibitedATX-mediatedLPA
activated, isolated platelets (Fulkerson et al., 2011). However,
the RGD sequence of the tandem SMB domains is not
involved in the binding of ATX to the b3 integrin of activated
formationbythrombin-
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platelets, as demonstrated by an ATX–RGE mutant that
showed a similar binding to activated platelets as that of
wild-type ATX (Hausmann et al., 2011). Although it has not
been tested directly, it is likely that LPA formation in blood
also depends on binding of ATX to the b3 integrins of acti-
vated platelets. In blood, ATX, which is present at a concen-
tration of approximately 100 nM in plasma (Nakamura et al.,
2008), has to compete with the much higher plasma concen-
tration of fibrinogen (5–8 mM) for binding to the b3 integrin
on activated platelets.
Healthy people display individual heterogeneity of the
LPA-induced platelet aggregation response when measured in
washed platelets, PRP or blood (Haseruck et al., 2004; Pamuk-
lar et al., 2008). However, all of the blood donors showed a
similar shape change response when tested in blood (Hase-
ruck et al., 2004). LPA-induced platelet aggregation was com-
pletely dependent on ADP-mediated activation of P2Y1 and
P2Y12receptors in whole blood (Haseruck et al., 2004). Thus,
LPA does not directly induce human platelet aggregation in
blood, as LPA-induced platelet aggregation requires the pres-
ence of extracellular ADP. In stirred blood, ADP may be either
secreted from platelet-dense granules or released from red
cells. In a recent study using isolated platelets, the individual
heterogeneityof LPA-induced
hypothesized to be due to LPA-induced activation of an
inhibitory pathway in the non-responders (Pamuklar et al.,
2008).
platelet aggregationwas
Low nM concentrations of LPA directly induce shape
change in washed platelets (Siess et al., 1999; Rother et al.,
2003). LPA binds to GPCRs on the platelet surface, and the
signal that is emitted by the activated platelet receptor is
transduced by the heterotrimeric G13protein to activate Rho
and Rho-kinase (Bauer et al., 1999; Klages et al., 1999; Grata-
cap et al., 2001; Moers et al., 2003). Rho-kinase phosphor-
ylatesthe130-kDmyosin-binding
phosphatase, thereby decreasing the activity of myosin phos-
phatase and increasing myosin light chain (MLC) phosphor-
ylation (Kimura et al., 1996; Bauer et al., 1999; Retzer and
Essler, 2000). Rho-kinase also phosphorylates and activates
LIM-kinase 1, which increases the turnover of phospho-
cofilin (Pandey et al., 2006; 2007). These biochemical path-
ways converge in the remodelling of actin–myosin structures
that underlie platelet shape change (Figure 1). Cytosolic Ca2+
increase, Rac activation and p21-activated kinase (PAK) are
not involved in LPA-induced shape change (Maschberger
et al., 2000; Pandey et al., 2007). High concentrations of LPA
(10 mM) induce an increase in cytosolic Ca2+in washed plate-
lets that is primarily due to the stimulation of Ca2+entry. This
leads to cofilin dephosphorylation and secretion; the latter
response requires, in addition, integrin aIIb3 outside-in sig-
nalling (Maschberger et al., 2000; Pandey et al., 2007). LPA
does not activate the heterotrimeric G-protein Giin platelets,
yet it shows a strong synergism in the induction of platelet
aggregation with platelet stimuli such as adrenaline and ADP
that activate Gi(Rother et al., 2003; Haseruck et al., 2004).
Until recently, the identity of the LPA receptor that medi-
ates platelet shape change remained obscure. Human plate-
lets express mRNA for the Edg receptors LPA1–3, and the
purinergic cluster LPA4 (GPR23), LPA5 (GPR92), LPA6 (P2Y5),
and the putative receptors GPR87 and P2Y10 (Amisten et al.,
2008; Khandoga et al., 2008; Pamuklar et al., 2008). The
receptors that are most abundantly expressed at the mRNA
level are LPA4and LPA5(Amisten et al., 2008; Khandoga et al.,
2008). A previous study suggested that LPA1and LPA3play a
role in inducing platelet shape change based on the ability of
the LPA receptor subtype-specific antagonist diacylglycerol
pyrophosphate to inhibit LPA-induced platelet shape change.
However, the inhibitory effect of this compound increased
with the time of pre-incubation (Rother et al., 2003), which is
not typical for a receptor antagonist. Two recent studies
described the involvement of LPA4 and LPA5 in mediating
LPA-induced platelet shape change (Khandoga et al., 2008;
Williams et al., 2009). However, firm evidence that these
receptors were the functional platelet LPA receptors was
lacking. The LPA response of platelets did not match the
pharmacological properties of the LPA4and LPA5receptors in
heterologous expression systems (Khandoga et al., 2008), and
the pharmacological receptor agonists and antagonists that
were used were not selective for LPA5(Williams et al., 2009).
More recently, it was demonstrated that LPA-induced
shape change in two human megakaryocytic cell lines was
inhibited by siRNA against LPA5, but not by knock-down of
each of the other receptors LPA1–4,6,7(Khandoga et al., 2011).
The rank order of activation by LPA species in these cells, with
alkyl-LPA 18:1 and alkyl-LPA 16:0 being the most potent, was
similar to that of human platelets, supporting the hypothesis
that LPA5 is the functional LPA receptor-mediating platelet
shape change (Figure 1). Importantly, siRNA against LPA5also
subunitofmyosin
Figure 1
LPA-induced platelet signalling during platelet shape change. Activa-
tion of the LPA5receptor coupled to the heterotrimeric G13 protein
stimulates Rho and Rho-kinase. The subsequent bifurcating pathway
directed to either the myosin-binding subunit of MLC phosphatase
or the LIM-kinase 1 leads to enhanced phosphorylation of MLC and
stimulation of phospho-cofilin turnover, respectively. Phosphorylated
myosin develops actin-activated ATPase activity, interacts with
F-actin, and assembles into filaments, whereas cofilin regulates actin
dynamics by enhancing both actin-polymerization and actin filament
severing. These cytoskeleton changes underlie the folding of the
surface membrane, the formation of pseudopods and the contractile
wave centralizing the secretory granules during platelet shape
change.
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inhibited shape change of the megakaryocytic cells that was
induced by the lipid-rich core of human atherosclerotic
plaques (Khandoga et al., 2011). Thus, LPA5 may be a novel
target for anti-thrombotic therapy for patients with ischae-
mic cardio- and cerebrovascular disease.
Upon plaque rupture, LPA exposed by the lipid core
might, by binding to LPA5, induce platelet shape change and
stimulate, in synergy with ADP, platelet aggregation and
thrombus formation (Haseruck et al., 2004; Rother et al.,
2003) (Figure 2). ADP can derive from damaged red cells
(Born and Wehmeier, 1979) and from activated platelets
adhering to the collagenous matrix of the ruptured cap and
secreting their granule contents (Penz et al., 2005; Reininger
et al., 2010) (Figure 2). Thus, plaque LPA may act as a cofactor
in acute atherothrombosis. However, the importance of LPA
in the lipid-rich core for platelet activation in whole blood
remains to be demonstrated. Specific LPA5 receptor antago-
nists will be helpful to answer this question. Indirect evidence
supports a role for plaque LPA in plaque-induced platelet
activation and thrombus formation. Low (sub-mM) concen-
trations of alkyl-LPA species, that may be reached locally after
plaque rupture, activate platelets in blood (Haseruck et al.,
2004), and aspirin is equally ineffective at inhibiting LPA-
triggered platelet aggregation in blood (Haseruck et al., 2004)
and plaque-induced platelet thrombus formation under arte-
rial flow conditions (Penz et al., 2007).
LPA in atherosclerosis
Hypercholesterolaemia promotes the continuous recruitment
of circulating monocytes to the arterial wall, which drives the
progression of atherosclerotic plaques (Swirski, 2006). LDL
enters the vessel wall and is retained in the subendothelial
space, where LDL is oxidatively modified. However, the type
and degree of LDL modifications are diverse, resulting in
distinct biological activities. Oxidized LDL is generally
divided into two main categories: minimally modified or
mildly oxidized LDL (MM–LDL or mox-LDL) and extensively
oxidized LDL (ox-LDL) (Stocker and Keaney, 2004; Levitan
et al.,2010). Althoughboth
MM–LDL differs from oxLDL, because it still binds to the LDL
receptor and is not recognized by most scavenger receptors
(Stocker and Keaney, 2004; Levitan et al., 2010). In contrast to
native LDL, MM-LDL can induce the adhesion of monocytes
to endothelial cells by up-regulating the CXC chemokine,
CXCL1, on the endothelial surface (Berliner et al., 1990;
Schwartz et al., 1994). Oxidized LDL or components of LDL
that are released during the oxidation, like oxidized phos-
pholipids, trigger the inflammatory response by activating
endothelial cells (Hansson and Hermansson, 2011; Weber
and Noels, 2011). Activated endothelial cells express adhe-
sion molecules and chemokines that direct the adhesion and
transmigration of circulating monocytes. These monocytes
arechemicallymodified,
Figure 2
Hypothetical role of plaque LPA and the platelet LPA5 receptor in acute atherothrombosis after plaque rupture. LPA in the lipid core of
atherosclerotic plaques may act as a cofactor with platelet-adhesive matrix proteins such as collagen type I and III in platelet activation (van Zanten
et al., 1994; Penz et al., 2005; Schulz et al., 2008). These matrix proteins are over-expressed in plaques as compared with healthy arterial intima.
LPA induces, through binding to the LPA5receptor, shape change of circulating platelets and has a synergistic effect with ADP at inducing platelet
aggregation and thrombus formation. ADP is secreted from dense granules of platelets adhering to the collagenous matrix of the ruptured cap.
GPVI, glycoprotein VI.
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transform into macrophages in the vessel wall, where they
engulf ox-LDL and propagate the inflammatory reaction in
concert with T-cells. During the progression of atherosclero-
sis, SMCs accumulate and form a fibrous cap that encloses a
highly thrombogenic core comprising extracellular lipids.
LPA accumulatesduring
perivascular collar placement in Apoe-/-mice and is increased
in the lipid core region of human atherosclerotic plaques
(Rother et al., 2003; Bot et al., 2010). In vitro, LPA has been
shown to activate NF-kB and increase the adhesion of mono-
cytestoendothelialcells
up-regulating adhesion molecules and chemokines, such as
intracellular adhesion molecule-1 (ICAM-1), E-selectin, and
vascular cell adhesion molecule-1 (VCAM-1) (Palmetshofer
et al., 1999; Rizza et al., 1999; Lee et al., 2004; Lin et al., 2007).
Whereas LPA-induced endothelial ICAM-1 expression is
mediated by the LPA1 receptor, both LPA1 and LPA3 are
involved in the chemotactic activity generated by the secre-
tion of IL-8 and chemokine ligand 2 (CCL2), also known
as monocyte chemotactic protein-1 (MCP-1) from LPA-
stimulated endothelial cells (Lee et al., 2004; Gustin et al.,
2008b). Additionally, the endothelial release of pentraxin-3 is
induced by LPA and enhances monocyte migration (Gustin
et al., 2008a). However, the role of LPA-induced endothelial
pentraxin-3 secretion in atherogenesis is unclear.
In murine carotid arteries, atherogenic monocyte adhe-
sion under flow conditions is primarily mediated by the CXC
chemokine CXCL1 (human GRO-a/murine KC), which is in
contrast to CCL2 immobilized on the endothelial surface
(Weber et al., 1999; Huo et al., 2001). Whereas most chemok-
ines are transcriptionally up-regulated, CXCL1 is stored in
intracellular vesicles of endothelial cells (Oynebraten, 2004;
Zhou et al., 2011). The modified LDL-induced secretion of
endothelial CXCL1 is mediated by an unsaturated LPA species
and requires the activity of ATX (Zhou et al., 2011). This
secretagogue effect of LPA is mediated by the LPA1/3-induced
activation of Rho-associated coiled-coil containing protein
kinase. However, activation of NF-kB by unsaturated LPA
atherogenesis inducedby
understatic conditions by
induces transcriptional up-regulation of CXCL1 in endothe-
lial cells, suggesting a biphasic response (Zhou et al., 2011).
This mechanism of LPA on CXCL1 is crucial in promoting
atherogenic monocyte recruitment and atherosclerosis in vivo
(Figure 3). Hyperlipidaemia-induced monocyte adhesion to
carotid arteries is almost completely abolished by pharmaco-
logical inhibition of LPA1/3, indicating that LPA is an impor-
tant mediator of the pro-atherogenic effects of ox-LDL (Zhou
et al., 2011). This reduced monocyte recruitment may explain
the inhibition of atherogenesis observed by blocking LPA1/3
receptors (Zhou et al., 2011).
Apart from the recruitment of monocytes, several cellular
functions of monocytes and macrophages, including the acti-
vation status, uptake of LDL and survival, play an important
role in atherogenesis (Moore and Tabas, 2011). Human
monocytes and macrophages primarily express LPA1 and
LPA2, and LPA has been shown to mediate the effects of
MM-LDL on monocyte activation via LPA1 (Fueller et al.,
2003; D’Aquilio et al., 2007). In agreement with this finding,
LPA stimulatestheexpression
cytokine IL-1b in a GPCR-dependent manner in a murine
macrophage cell line (Chang et al., 2008b). Moreover, LPA
increases the uptake of ox-LDL in monocytes/macrophages
(Llodra et al., 2004; Chang et al., 2008b). In J774A murine
macrophages, which express LPA2 and LPA3, LPA-induced
lipid accumulation is mediated by the up-regulation of the
scavenger receptor A and can be inhibited by the LPA1/LPA3
receptor antagonist Ki16425 (Chang et al., 2008a). In addi-
tion, LPA binds to the nuclear receptor PPARg and induces
PPARg-dependent CD36 expression in murine macrophages
(McIntyre et al., 2003). PPARg activation in macrophages has
anti-inflammatory effects, and conditional deletion of PPARg
in macrophages enhances atherosclerosis (Babaev et al., 2005;
Bouhlel et al., 2007). Therefore, an atheroprotective role of
LPA by activation of PPARg could be envisioned, if this
mechanism is relevant in vivo. Although LPA is known to
increase monocyte migration (Zhou et al., 1995; Gustin et al.,
2008b), inhibition of reverse transmigration of monocytes by
of thepro-atherogenic
Figure 3
LPA promotes the accumulation of macrophages in atherosclerotic lesions. The moxLDL leads to increased formation of LPC, which is converted
by endothelial-derived ATX into LPA. LPA triggers the release of the chemokine CXCL1 from endothelial cells through the activation of LPA1and
LPA3. CXCL1 is immobilized on the endothelial surface and induces the adhesion of monocytes to the vessel wall via its receptor CXCR2 on
monocytes. These monocytes migrate into the subendothelial space and transform into macrophages, which are the primary cells in early
atherosclerotic plaques.
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LPA has been found, which may play a role in the regression
of atherosclerosis (Llodra et al., 2004). Moreover, LPA protects
macrophages from apoptosis by activating phosphoinositide
3-kinase (PI3K), indicating that LPA impairs the removal of
plaque macrophages (Koh et al., 1998).
SMCs accumulate in advanced atherosclerotic plaques,
and apoptosis of SMCs is associated with accelerated athero-
sclerosis. However, exacerbated proliferation of SMCs and
the conversiontoapro-inflammatory
type may promote the progression of atherosclerosis by
enhanced monocyte recruitment (Zeiffer et al., 2004). LPA
potently stimulates the proliferation of cultured SMCs and
most probably contributes to ox-LDL-induced SMC prolif-
eration (Tokumura et al., 1994; Natarajan et al., 1995; Kim
et al., 2006; Damirin et al., 2007; Komachi et al., 2009). LPA
mediates the MM–LDL-induced expression of CCL20 in
SMCs via LPA receptors, which is secreted from atheroscle-
rotic plaques and elevated in the circulation of patients
with hyperlipidaemia (Calvayrac et al., 2011). However, the
functional role of LPA-induced CCL20 in atherogenesis
needs to be determined. Tissue factor (TF) is a critical deter-
minant of atherosclerotic plaque thrombogenicity. LPA
up-regulates TF expression in SMCs via Gi-protein–mediated
activation of ERK1/2 (Cui et al., 2003). Furthermore, LPA
induces the pro-atherogenic factors CCL2 (MCP-1) and IL-6
in human vascular SMCs in vitro (Kaneyuki et al., 2007; Hao
et al., 2010).
Taken together, lipoprotein-derived LPA plays a crucial
role in atherogenesis by promoting the recruitment of mono-
cytes. Therapeutic targeting of LPA1 or LPA3 on endothelial
cells may be a promising approach against atherosclerosis.
SMCpheno-
LPA in vascular remodelling
The arterial vessel wall can adapt to variety of environmental
cues, such as changes in pressure, flow, or oxygen supply, by
a process called vascular remodelling that mainly includes a
response of the SMCs and results in structural alterations of
the tunica media (Gibbons and Dzau, 1994). The media may
enlarge, for example in arterial or pulmonary hypertension,
or diminish, as in the formation of aneurysms. Furthermore,
neointimal accummulation of SMCs is characteristic of vas-
cular repair after injury, for example following stent implan-
tation into atherosclerotic arteries (called restenosis). In
addition, endothelial cells and inflammatory cells like
monocytes/macrophages affect SMC function and the pro-
duction of extracellular matrix proteins, thereby playing an
important role in vascular remodelling (Schober, 2008). The
phenotypic switch of SMCs from a contractile, quiescent state
towards a synthetic, proliferative state is a common feature
during vascular remodelling and may be causally related to
neointima formation (Owens et al., 2004).
Both migration and proliferation of medial SMCs follow-
ing vascular injury have been implicated in neointima for-
mation (Schwartz et al., 1995). In vitro, LPA has been shown to
induce the proliferation of SMCs via activation of LPA1 and
Gi/q proteins and involves PKC, ERK1/2, the PI3K/PKB (Akt)
pathway and MAPK cascades (Tokumura et al., 1994; Seewald
et al., 1997; 1999; Gennero et al., 1999; Schmitz et al., 2002;
Xu et al., 2003; Gouni-Berthold et al., 2004; Baldini et al.,
2005; Kim et al., 2006; Komachi et al., 2009). In contrast,
combined genetic deletion of LPA1 and LPA2 is required to
inhibit serum-induced growth of SMCs (Panchatcharam
et al., 2008). Furthermore, LPA enhances migratory activity of
vascular SMCsbyGi/q
protein-coupled
mediated activation of the p38MAPK pathway (Kim et al.,
2006; Damirin et al., 2007; Komachi et al., 2009; Zhou et al.,
2009). However, SMCs from LPA1-/-mice showed increased
migration due to compensatory up-regulation of LPA3 (Pan-
chatcharam et al., 2008). Whereas the migration of LPA2-
deficient SMCs is not impaired, combined deletion of LPA1
and LPA2 inhibits SMC migration (Panchatcharam et al.,
2008). These results indicate that LPA3 also promotes SMC
migration in the presence of LPA2. Neointimal SMCs display
a pro-inflammatory phenotype driven by activated NF-kB
signalling, which may promote leucocyte recuitment to the
injured artery (Zeiffer et al., 2004). LPA increases the expres-
sion of IL-6 and CCL2 in vascular SMCs via LPA1 and
PKC-mediated p38 MAPK and NADPH oxidase-dependent
generation of reactive oxygen species, respectively (Kaneyuki
et al., 2007; Hao et al., 2010). The early growth response gene
1 (Egr1) is crucial for SMC proliferation and neointima for-
mation (Khachigian, 2006). Egr1 is induced by LPA-mediated
activation of the transcription factors cAMP response
element-binding (CREB) and serum response factor (SRF) in
SMCs (Cui et al., 2006). However, the role of LPA-induced
Egr1 in vascular remodelling remains to be defined. The
reduced expression of contractile proteins is characteristic for
synthetic SMCs, and this dedifferentiation process has been
implicated in neointima formation (Owens et al., 2004). LPA
species with an unsaturated fatty acyl chain selectively down-
regulate contractile protein expression in SMCs via sustained
activation of ERK1/2 and p38 MAPK (Hayashi et al., 2001).
This effect of LPA on SMC dedifferentiation has been attrib-
uted to the activation of LPA3 using LPA receptor-specific
antagonists and agonists (Zhou et al., 2010). In agreement
with this finding, no differences in LPA-induced SMC dedif-
ferentiation have been observed between wild-type, LPA1-/-,
LPA2-/-, LPA1-/-/LPA2-/-and PPARg-/-SMCs (Guo et al., 2008;
Panchatcharam et al., 2008). However, the PPARg agonist ros-
iglitazone reduced the expression of SMC markers, like
unsaturated LPA, indicating different pathways for PPARg-
and LPA-induced SMC differentiation (Zhang et al., 2004).
Taken together, LPA acts in multiple ways on SMCs in vitro,
which suggests that LPA promotes vascular remodelling and
neointima formation.
To study the role of LPA in vascular remodelling in vivo,
short-term incubation of the carotid arteries with LPA has
been performed. Intriguingly, transient treatment only with
unsaturated LPAs, like LPA18:1, LPA20:4 or the LPA analogue
1-AGP18:1 (1-O-octadecenyl glycerophosphate), triggers the
formation within weeks of a neointima that primarily con-
sists of SMCs (Yoshida et al., 2003; Zhang et al., 2004; Cheng
et al., 2009; Subramanian et al., 2010). Blocking LPA18:1-
induced ERK and p38MAPK activation prevented neointima
formation (Yoshida et al., 2003). Furthermore, LPA20:4 and
1-AGP18:1-induced neointima formation was reduced by
treatment with a PPARg antagonist and in mice with PPARg-
deficiency in vascular wall cells (Zhang et al., 2004; Cheng
et al., 2009). Although LPA20:4-induced neointima formation
was partially inhibited by pertussis toxin and the LPA1/LPA3
LPA1
receptor-
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antagonist dioctylglycerol pyrophosphate in rats, which do
not express LPA3 in the carotid wall, neointimal growth fol-
lowing 1-AGP18:1 incubation was not impaired in LPA1-/-,
LPA2-/-and LPA1-/-/LPA2-/-mice (Zhang et al., 2004; Cheng
et al., 2009). Neointima formation by carotid ligation is
increased in LPA1-/-mice, whereas a combined deficiency of
LPA1 and LPA2 diminishes neointimal growth (Panchatch-
aram et al., 2008). The compensatory overexpression of LPA3
in SMCs from LPA1-/-mice, which is absent in LPA1-/-/LPA2-/-
SMCs, is associated with enhanced neointima formation
(Panchatcharam et al., 2008). PPARg activation in SMCs has
been shown to inhibit proliferation and migration and
attenuates neointimal hyperplasia after vascular injury (Lim
et al., 2006; Lee et al., 2009; Zhang et al., 2011). Accordingly,
1-AGP18:1, but not rosiglitazone, increases injury-induced
neointima formation, indicating that LPA-mediated PPARg
activation does not play a role in vascular remodelling fol-
lowing vascular injury (Cheng et al., 2009). Taken together,
these results show that molecular mechanisms of LPA-
dependent neointima formation may differ between local
treatment with LPA and in established animal models of
vascular remodelling.
In atherosclerosis-prone Apoe-/-mice, treatment with the
LPA1/LPA3antagonist Ki16425 diminished neointimal hyper-
plasia after carotid wire injury by reducing the SMC and
macrophage content in the lesions, indicating that LPA pro-
motes injury-induced neointima formation via LPA receptors
(Subramanian et al., 2010). Interestingly, unsaturated but
not saturated LPA induces sustained up-regulation of the
chemokine CXCL12 (stromal cell-derived factor 1) in the
vessel wall, which mobilizes and recruits smooth muscle pro-
genitor cells (SPCs) from the bone marrow into the injured
artery (Schober, 2008; Subramanian et al., 2010) (Figure 4).
Silencing of either LPA1 or LPA3 in the vessel wall impaired
the LPA20:4-induced neointima formation and SPC mobili-
zation, suggesting that, similar to atherogenic monocyte
recruitment, these two LPA receptors independently trigger
the vascular response (Subramanian et al., 2010). It remains
to be determined whether this functional independency
might be due to the formation of heterodimers between LPA1
and LPA3, and whether LPA1/LPA3 heterodimers have an
increased binding affinity to unsaturated LPAs (Zaslavsky
et al., 2006). The different mechanisms that were observed
in LPA-induced neointimal hyperplasia might be dose-
dependent. Whereas 40 mM of LPA20:4 has been found to
trigger CXCL12 expression, unsaturated LPAs in a dosage
range between 1 and 10 mM induce ERK1/2/p38MAPK- or
PPARg–dependent neointima formation (Yoshida et al., 2003;
Zhang et al., 2004; Cheng et al., 2009; Subramanian et al.,
2010). However, the fact that pharmacological inhibition of
LPA1 and LPA3 after carotid injury inhibits CXCL12 expres-
sion and impairs CXCL12-dependent SPC mobilization indi-
cates that the 40 mM LPA20:4 dose used for local carotid
treatment closely mirrors the role of LPA after vascular injury
(Subramanian et al., 2010). Microvesicles released from
apoptotic SMCs following injury have been found to
up-regulate CXCL12 expression in non-injured SMCs, and
blocking the apoptosis of SMCs following vascular injury
greatly inhibits CXCL12 expression (Zernecke et al., 2005).
Taking into account the role of microvesicles in LPA genera-
tion (Fourcade et al., 1995), increased LPA production due to
the release of microvesicles from apoptotic SMCs may con-
tribute to the up-regulation of CXCL12 in the injured arter-
ies. In addition, activated platelets adhering to the denudated
vascular surface may not only present CXCL12 during the
recruitment of SPCs, but also increase CXCL12 expression by
enhancing LPA formation (Zernecke et al., 2005).
Remodelling of the pulmonary vasculature occurs during
the hypoxia-driven development of pulmonary hypertension
and is characterized by the thickening of the media and
adventitia in pulmonary arteries and muscularization of non-
muscular alveolar wall vessels (Stenmark et al., 2006). Inter-
estingly, hypoxia-induced pulmonary hypertension and
remodelling is accelerated in both ATX+/-mice and LPA1-/-/
LPA2-/-mice (Cheng et al., 2012). In LPA1-/-/LPA2-/-mice,
thickening of the arteriolar vessel wall is already evident
during normoxia, and aging results in severe pulmonary
hypertension (Cheng et al., 2012). This increased remodelling
of pulmonary vessels was associated with increased expres-
sion of genes involved in endothelin-1 signalling (Cheng
Figure 4
LPA induces neointima formation after vascular injury. Following vascular injury, activated platelets adhere to the denuded surface of the vessel
wall and medial SMCs undergo apoptosis. These early events after vascular injury may induce the production of LPA, which increases CXCL12 in
the vessel wall through its receptors LPA1and LPA3. CXCL12 is released into the circulation and recruits SPC via its receptor CXCR4 to the injury
site. These SPCs differentiate into neointimal SMCs, which form the neointimal lesion.
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LPA in atherosclerosis
British Journal of Pharmacology (2012) 167 465–482475

Page 12

et al., 2012). In contrast, the deficiency of either LPA1or LPA2
is not associated with pulmonary hypertension. This indi-
cates that LPA signalling via LPA1and LPA2in the pulmonary
vasculature has an important homeostatic role and appears
to have protective effects in hypoxia-induced pulmonary
hypertension.
Conclusions and future directions
LPA has atherogenic actions, and specific LPA receptors have
been identified that may mediate some of these harmful LPA
activities. The experimental evidence is based not only on
numerous in vitro studies, but also on several in vivo studies.
LPA does not only enhance atherosclerosis, but also vice versa,
cardiovascular risk factors affect the ATX/LPA axis thereby
further aggravating the progression of atherosclerotic dis-
eases. Obesity and hyperlipidaemia enhance ATX expression
and activity, and the generation of LPA in the circulation.
Increased ATX-dependent LPA formation from LDL-derived
LPC promotes endothelial chemokine (CXCL1) secretion
leading to the recruitment of monocytes to the atheroscle-
rotic lesion, and thereby enhancing atherosclerosis (Figure 3).
Endothelial LPA1/3receptors and Rho-kinase are critical here.
LPA also increases the uptake of ox-LDL in monocytes/
macrophages by up-regulation of the scavenger receptor A,
and inhibits monocyte egression from plaques. LPA may also
induce these effects by binding to LPA1/3 and to the nuclear
receptor PPARg. LPA also stimulates VSMC dedifferentiation,
proliferation and neointima formation. Also here, LPA1 and
LPA3 receptors as well as PPARg seem to be involved; the
situation is, however, complex, as the mechanism of LPA-
dependent neointima formation seems to differ between
local treatment with LPA and established animal models of
vascular remodelling. Lastly but not least, LPA accumulating
in the lipid-rich core may contribute to acute thrombus for-
mation after plaque rupture by stimulation of the platelet
LPA5 receptor and Rho-kinase (Figures 1, 2). Future studies
might be directed to explore the role of the non-Edg recep-
tor family of LPA receptors in various animal models of
atherosclerosis, to determine the expression of individual LPA
receptors in human atherosclerotic lesions in situ and to
evaluate the effects of novel LPA receptor antagonists on the
development, progression and regression of atherosclerosis in
animal models, as well as on thrombosis induced by athero-
sclerotic plaques in human models. Clinical studies could
determine whether plasma levels of defined LPA molecular
species could be a novel biomarker of cardiovascular risk. In
such studies, the addition of pharmacological ATX inhibitors
to the blood collection tube might be important to minimize
artificial in vitro LPA formation during blood handling.
Acknowledgements
This publication was made possible by grants to AS and WS
from the Deutsche Forschungsgemeinschaft (Scho 1056/2,
Scho 1056/3, Si 274/9; Si 274/11), the August-Lenz-Stiftung
and the Bayern University.
Statement of conflict of interest
The authors have no conflict of interest.
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BJP
LPA in atherosclerosis
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