Transactivation of Flk-1/KDR by lysophosphatidylcholine induces vascular endothelial cell proliferation

Full text

Page 1

Title:
Transactivation of Flk-1/KDR by lysophosphatidylcholine induces vascular endothelial cell
proliferation
Yoshiko Fujita1), Masanori Yoshizumi1) Yuki Izawa1), Nermin Ali1), Hideki Ohnishi1), Yasuhisa
Kanematsu1), Keisuke Ishizawa1), Koichiro Tsuchiya2), Toshiaki Tamaki1)
Department of Pharmacology1), Graduate School of Medical Sciences and Department of
Clinical Pharmacology2), Graduate School of Pharmaceutical Sciences, The University of
Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
Abbreviated title:
LPC transactivates Flk-1/KDR in HUVEC
Corresponding Author:
Yoshiko Fujita
Department of Pharmacology, The University of Tokushima Graduate School of Medical
Sciences, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
Telephone - (81) -886-33-7061
FAX - (81) -886-33-7062
e-mail: fujita56@ri.ncvc.go.jp
Key words: LPC, c-Src, endothelial cell
1

Endocrinology. First published December 1, 2005 as doi:10.1210/en.2005-0644
Copyright (C) 2005 by The Endocrine Society

Page 2

ABSTRACT
Lysophosphatidylcholine (LPC), a major lipid component of oxidized low density lipoprotein
(oxLDL), is a bioactive lipid molecule involved in numerous biological processes including the
progression of atherosclerosis. Recently, orphan G-protein-coupled receptors (GPCRs) were
identified as high-affinity receptors for LPC. Although several GPCR ligands transactivate
receptor tyrosine kinases (RTKs), LPC-stimulated transactivation of RTK has not yet been
reported. Here we observed for the first time that LPC treatment of human umbilical vein
endothelial cells (HUVEC) induces tyrosyl phosphorylation of vascular endothelial growth factor
(VEGF) receptor 2 (fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR).
Flk-1/KDR transactivation by LPC was inhibited by VEGF receptor tyrosine kinase inhibitors,
SU1498 and VTKi in immunoprecipitation. Furthermore, we examined the effects of the Src
family kinases inhibitors, Herbimycin A and PP2 on LPC-induced Flk-1/KDR transactivation.
Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation
because Herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src
transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from
Western blots, extracellular signal-regulated kinase 1/2 and Akt, which are downstream effectors
of Flk-1/KDR, were also activated by LPC and this was inhibited by SU1498, VTKi,
Herbimycin A, PP2 and KI Src transfection in HUVEC. LPC-induced stimulation of HUVEC
proliferation was shown to be secondary to transactivation because it was suppressed by SU1498,
VTKi, Herbimycin A, PP2 and KI Src transfection in MTT assay. These findings suggest that
LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the
progression of atherosclerosis.
2

Page 3

Introduction
Lysophosphatidylcholine (LPC) is known to induce a variety of vascular endothelial
responses ranging from the upregulation of adhesion molecules and growth factors to the
secretion of chemokines and superoxide anion radicals (1, 2). As a component of oxidized low
density lipoprotein (oxLDL), LPC is locally generated and accumulates at the sites of wounds,
inflammation and atherosclerosis (3, 4). Atherosclerosis can be characterized as a chronic
inflammatory disease in which both cell proliferation and apoptotic/necrotic cell death occur
within the vascular wall. In atherosclerotic lesions, pathological examination reveals the
presence of angiogenesis (5, 6). It has previously been shown that LPC induces growth factor
gene expression in cultured human endothelial cells (7). It has also been shown that oxLDL and
LPC induces endothelial proliferation (8).
Recently, orphan G-protein-coupled receptors (GPCRs) were identified as high-affinity
receptors for LPC. LPC binds to G2A and GPR4, Gi-protein-coupled receptors, specifically and
regulates both cell growth and immunologic responses (9, 10). Gs-protein-coupled receptor
GPR119 is reported to be a novel LPC receptor involved in insulin secretion (11). LPC,
interacting with its receptor, induces an elevation of Ca2+ concentration activates serum
responsive transcription factors via the mitogen-activated protein kinase (MAPK) pathway (10,
12). In spite of the continued accumulation of evidence, however, the exact mechanisms by
which LPC exerts biological function in endothelial cells (EC) have not yet been elucidated.
Transactivation of receptor tyrosine kinases (RTKs) by the binding of ligands to GPCRs has
been shown to have important physiological consequences. GPCR-mediated RTK
transactivation has been implicated to have a crucial role in diseases such as cardiac hypertrophy
and cancer, and may have an important role in vascular diseases as well (13, 14). Role of Ca2+,
3

Page 4

reactive oxygen species, cSrc, Pyk2, protein kinase C and membrane-bound metalloproteases has
been reported in GPCR-mediated transactivation of RTKs (15-18). Among these intracellular
and extracellular molecules, a role for c-Src tyrosine kinase in GPCR-mediated RTK
transactivation has been the focus (16, 19). LPC has been reported as a regulator of tyrosine
kinase activity (20, 21). In these investigations, c-Src is suggested to have an important role in
LPC signaling. It has been reported that transactivation of RTK in response to stimulation of
GPCRs induces MAPK and Akt activation (15, 19). Vascular endothelial growth factor receptor-
2 (fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR) is a major receptor
tyrosine kinase transducing the effects of VEGF into EC. The signaling of Flk-1/KDR is
necessary for the execution of VEGF-stimulated proliferation as well as the survival of cultured
EC, and has been shown to be involved in atherosclerosis. Therefore, we hypothesized that LPC
may transactivate Flk-1/KDR and investigated the specific role of LPC-induced Flk-1/KDR
activation in EC.
In the present study, we examined whether LPC transactivates Flk-1/KDR in human
umbilical vein endothelial cells (HUVEC). Thereafter, we investigated the involvement of c-Src
tyrosine kinase in Flk-1/KDR transactivation by LPC. The findings of the present study strongly
suggest that the transactivation of Flk-1/KDR by LPC is mediated by c-Src in HUVEC. The
influence of Flk-1/KDR transactivation on the activation of extracellular signal-regulated kinase
(ERK) 1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also examined. In
addition, it was observed that LPC caused HUVEC proliferation, which may be related to the
progression of atherosclerosis.
4

Page 5

Materials and Methods
Chemicals
LPC (Lysophosphatidylcholine), (C18:0) and wortmannin were purchased from Sigma (St.
Louis, MO). Recombinant human VEGF was from PepRo Tech EC, Ltd. (London, UK). VEGF
receptor tyrosine kinase inhibitors VTKi (4-[(4'-chrolo-2'-fluoro) phenylamino]-6,7-
dimethoxyquinazoline) and SU1498 ((E)-3-(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-
propyl) amino-carbonyl] acrylonitrile), and Src family tyrosine kinase inhibitors PP2 (4-amino-
5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine) and Herbimycin A were from
Calbiochem (San Diego, CA). MEK1/2 inhibitors PD98059 was from WakoPure Chemical
Industries, Ltd. (Osaka, Japan) and U0126 was from Promega (Madison, WI). anti-Flk-1/KDR
polyclonal antibody, protein A/G PLUS-agarose beads and anti-ERK1/2 antibody were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine, clone 4G10 and Anti-
Src antibody were from Upstate Biotechnology Inc. (Lake Placid,NY). Anti-phospho-ERK1/2
(Thr202/Tyr204) antibody, anti-phospho-Akt (Ser473) antibody and anti-Akt antibody were from
Cell Signaling Technology Inc. (Beverly, MA). Anti-Src phosphospecific antibody (Tyr418),
which recognizes the activated form of c-Src, was from Biosource (Camarillo, CA). All other
chemicals were of reagent grade, were obtained from commercial sources, and were used
without further purification.
Cell culture and transient transfection
HUVEC and endothelial cell basal medium-2 (EBM-2) were purchased from Clonetics (San
Diego, CA). HUVEC were cultured in EBM-2 supplemented with 10 % fetal bovine serum
(FBS), gentamicin sulphate (50 ?g/ml), amphotericin-B (50 ng/ml), in addition to human
5

Page 6

fibroblast growth factor-B (hFGF-B; 10 ng/ml), epidermal growth factor human recombinant in a
buffered BSA saline solution (hEGF; 20 ng/ml), vascular endothelial growth factor human
recombinant (hVEGF; 1 ng/ml), insulin-like growth factor-1 in aqueous solution cell culture
tested (IGF-1; 1 ng/ml), ascorbic acid (1 ?g/ml), heparin (3 ng/ml), hydrocortisone (0.4 ?g/ml) at
37 ?C and 5 % CO2. For transfection of wild-type (WT) or kinase-inactive (KI) Src,
commercially available pUSE mammalian expression vectors encording pp60c-Src or
catalytically-inactive Src (K297R) were used (Upstate Biotechnology, Inc.). For the transient
expression experiments, HUVEC were transfected with CytoPure-huv Transfection Reagent
(polyplus-transfection, QBIOGENE Inc., Carlsbad, CA) according to the manufacture's
instructions. Transfection efficiency was determined with pcDNA3.1-GFP transfection as about
30 % in HUVEC.
Immunoblot analysis
HUVEC in 0.2 % fetal bovine serum (FBS)-containing EBM-2 medium were treated with or
without inhibitors for various times, and then incubated with LPC. After treatment with
reagents, the cells were washed twice with cold phosphate buffered saline. Thereafter, the cells
were harvested using 0.5 ml of lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM
Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ?-
glycerophophosphate, 1 mM sodium orthovanadate, 1 ?g/ml leupeptin, 1 mM
phenylmethylsulfonylfluoride) and incubated on ice. After thawing, cells were harvested and
cell lysates were sonicated (Handy Sonic UR-20 P, Tomy Seiko Co, Ltd., Tokyo, Japan) on ice
for 15 s, and then centrifuged at 25,000 x g for 20 min at 4 ?C to precipetate cell debris. The
supernatant of lysates was analyzed for protein concentration by the Bradford methods (Bio-Rad,
6

Page 7

Hercules, CA), and equal amounts of cellular proteins were separated by SDS-PAGE. After
transfer to nitrocellulose membrane, the activation levels of ERK1/2, Akt and Src were examined
using anti-phosphospecific antibodies, as described previously (22, 23).
Immunoprecipitation
Lysates containing equal amounts of protein were incubated with anti-Flk-1/KDR antibody
overnight and then incubated with protein A/G PLUS-agarose beads for 2 h on a roller system at
4 ?C. After immunoprecipitation, samples were evaluated with immunoblot assay using anti-
phosphotyrosine antibody (clone 4G10). Based on its 230-kD molecular weight and tyrosyl
phosphorylation, it was considered that the bands represent activated state of Flk-1/KDR as
described previously (17).
Cell viability assay
When HUVEC reached 40-50 % confluence in 35-mm collagen-coated dishes (IWAKI,
Osaka Japan), growth was arrested using EBM-2 medium with 0.2 % FBS for 24 h. SU1498,
VTKi, PD98059, U0126 and Herbimycin A were added and incubated for 30 min. PP2 was
added and incubated for 2 h. Following 24 h incubation with LPC (20 ?M), MTT was added at a
final concentration of 0.5 mg/ml, and after a further 2 h incubation, HUVEC were lysed with
isopropanol containing 0.04 M HCl. MTT reduction was read at 550-nm by a
spectrophotometer.
Statistical analysis
7

Page 8

Values are presented as means ? S.D. for 5 separate experiments. One-way analysis of
variance was used to determine significance among groups, after which post-hoc test with the
Bonferroni correction were used for comparison between individual groups. A value at P<0.05
was considered to be significant.
8

Page 9

Results
LPC stimulates transactivation of Flk-1/KDR in HUVEC. We first examined whether
Flk-1/KDR is activated by LPC in HUVEC. HUVEC were treated at various times and with
various concentrations of LPC. Activation of Flk-1/KDR in the cell lysates was determined by
tyrosyl phosphorylation as described in Experimental Procedures. As shown in Fig. 1A, LPC
rapidly activated Flk-1/KDR (with a peak at 10 min) and then sustained activation for at least 60
min. Fig. 1B shows the dose response for the activation of Flk-1/KDR by LPC in HUVEC. Flk-
1/KDR activation was determined by a 10 min incubation period. Flk-1/KDR activity was
increased in a dose-dependent manner by LPC (1 ?M to 20 ?M), and maximal activation
occurred at 20 ?M of LPC. To confirm that these are transactivation events, we examined the
effects of VEGF receptor tyrosine kinase inhibitors, SU1498 (30 ?M) and VTKi (5 ?M) on LPC-
induced Flk-1/KDR activation in HUVEC. The cells were pretreated with SU1498 and VTKi for
30 min before the addition of LPC (20 ?M) and VEGF (10 ng/ml) for 10 min. As shown in Fig.
2A, both compounds significantly inhibited phosphorylation of Flk-1/KDR in response to 20 ?M
LPC. Fig 2B shows that both compounds also inhibited 10 ng/ml VEGF-induced
phosphorylation of Flk-1/KDR. There were no differences in the total amounts of Flk-1/KDR
observed on Western blot analysis with anti-Flk-1/KDR antibodies. These findings suggest that
LPC significantly transactivates Flk-1/KDR in HUVEC.
c-Src tyrosine kinase is involved in the LPC-induced Flk-1/KDR transactivation.
Many tyrosine kinases, including those of both the receptor and non receptor type, are important
for the activation of survival and/or proliferative pathways in various cell types. Several c-Src-
mediated signal transduction pathways from GPCRs to receptor tyrosine kinases have been
9

Page 10

reported (17, 18). We hypothesized that c-Src might be involved in the LPC-induced
transactivation of Flk-1/KDR. To elucidate whether LPC activates c-Src in HUVEC, an
examination of the effect of LPC on c-Src tyrosine kinase activity in HUVEC was undertaken
As shown in Fig. 3A, an application of 20 ?M LPC caused rapid and significant phosphorylation
of c-Src (peak at 5 min) at tyrosine 418, an autophosphorylation cite that leads to full catalytic
activity of the kinase. c-Src activity was increased by LPC in a dose-dependent manner (1 ?M to
20 ?M), and maximal activation occurred at 20 ?M of LPC (Fig. 3B). A tyrosine kinase
inhibitor, Herbimycin A (1 ?M), and a Src kinase family inhibitor, PP2 (10 ?M), both inhibite
LPC-induced Src kinase activation (data not shown). In addition, Herbimycin A (0.1 ?M to 10
?M) and PP2 (1 ?M to 100 ?M) both inhibited LPC-induced Flk-1/KDR activation in a dose-
dependent manner (Fig. 4A, B). The cells were transfected with KI Src for 24 h and were
pretreated with PP2 for 2 h before the addition of LPC (20 ?M) for 10 min. As shown in Fig. 4
KI Src transfection and PP2 (100 ?M) treatment did not affect VEGF-induced Flk-1/KDR
activation. Furthermore, we examined the effect of KI Src transfection on LPC-induced Flk-
1/KDR activation. As shown in Fig. 5A, transfection of KI Src almost abolished LPC-induced
Flk-1/KDR activation, while WT Src transfection caused Flk-1/KDR activation with or whitout
LPC treatment. Transfection of KI Src also inhibited LPC-induced c-Src kinase activation in
HUVEC (Fig. 5B). There were no differences in the total amounts of Flk-1/KDR observed on
Western blot analysis with anti-Flk-1/KDR antibody. In WT Src and KI Src cDNA transfected
samples, c-Src protein expression were different from vector alone transfected sample. These
findings strongly suggest that c-Src is involved in LPC-induced Flk-1
.
d
C,
/KDR transactivation in
HUVEC.
10

Page 11

LPC stimulates downstream of Flk-1/KDR and c-Src. It was reported that ERK1/2 and
Akt exist in the downstream region of Flk-1/KDR (24). It has also been reported that LPC
stimulates ERK1/2 and Akt activation which results in the activation of cell survival pathw
(25). Thus, we examined whether LPC activates ERK1/2 and Akt through transactivation of Flk-
1/KDR in HUVEC. Activation of ERK1/2 and Akt in the cell lysates were determined as
described in Experimental Procedures. Application of 20 ?M LPC caused significant activat
of ERK1/2 which peaked at 60 min, and also of Akt, which peaked at 90 min (data not show
First, we investigated whether LPC-induced activation of ERK1/2 and Akt was inhibited by
SU1498 (30 ?M) or VTKi (5 ?M) in HUVEC. The cells were pretreated with SU1498 and
VTKi for 30 min before the addition of LPC (20 ?M), to examine their effects on ERK1
activation, respectively. Both compounds significantly inhibited ERK1/2 and Akt activation
response to 20 ?M LPC stimulation (Fig. 6). These findings suggest that LPC-induced
transactivation of Flk-1/KDR is involved in the activation of ERK1/2 and Akt downstream.
Furthermore, we examined the effects of Herbimycin A, PP2 and KI Src transfection on ERK1/2
and Akt activation by LPC to investigate whether c-Src is involved in these phenomena. The
cells were pretreated with Herbimycin A (1 ?M) for 30 min and PP2 (10 ?M) for 2 h before the
addition of LPC (20 ?M), to examine their respective effects on ERK1/2 or Akt activation.
Herbimycin A and PP2 both inhibited LPC-induced ERK1/2 and Akt phosphorylation (Fig. 7A
B). Transfection of KI Src also inhibited LPC-induced ERK1/2 and Akt phosphorylation in
HUVEC (Fig. 7C, D
ays
ion
n).
/2 or Akt
in
,
). These findings suggest that ERK1/2 and Akt are downstream effector
molecules of c-Src, which is suggested to be involved in the LPC-induction of Flk-1/KDR
transactivation.
11

Page 12

LPC-induced Flk-1/KDR transactivation stimulates HUVEC proliferation. Flk-
activation is reported to induce cell growth and differentiation (24). Therefore, to investiga
pathophysiological implications of LPC-induced Flk-1/KDR transactivation, we examined the
effect of LPC on HUVEC proliferation. As shown in Fig. 8A, MTT assay revealed that
stimulation with LPC increased HUVEC viability over a 24 h incubation period in a dose-
dependent manner (1 ?M to 20 ?M). In addition, we examined whether LPC-induced HUVEC
proliferation was mediated by Flk-1/KDR transactivation as well as activation of ERK1/2 and
Akt. The cells were pretreated with SU1498 (30 ?M), VTKi (5 ?M), the MEK1/2 inhibitors
PD98059 (10 ?M) and U0126 (10 ?M), and the phosphatidylinositol 3-phosphate kinase (PI3
inhibitor LY294002 (10 ?M) and Wortmannin (10nM) for 30 min before the addition of L
?M). SU1498, VTKi, PD98059, U0126, LY294002 and Wortmannin inhibited LPC-induced
HUVEC proliferation (Fig. 8B). These results suggest that ERK1/2 and Akt are involved in
LPC-induced proliferation of HUVEC. To characterize the role of c-Src in LPC-induced
HUVEC proliferation, we then examined the effect of Herbimycin A and PP2 on LPC-induc
HUVEC proliferation. The cells were pretreated with Herbimycin A (1 ?M) for 30 min and P
(10 ?M) for 2 h before the addition of LPC (20 ?M). Both Herbimycin A and PP2 significantly
inhibited the HUVEC proliferation induced by LPC (Fig. 8C). Transfection of KI Src also
inhibited LPC-indu
1/KDR
te
K)
PC (20
ed
P2
ced HUVEC proliferation whereas WT Src transfection induced HUVEC
proliferation in the absence of LPC stimulation (Fig. 8D). These findings strongly suggest that
c-Src is involved in LPC-induced HUVEC proliferation, which is mediated through Flk-1/KDR
transactivation.
12

Page 13

Discussion
The major finding of the present study is that LPC transactivates VEGF receptor Flk-1/KDR
in HUVEC via c-Src tyrosine kinase activation. SU1498 and VTKi, which have been shown to
inhibit Flk-1/KDR, inhibited LPC-induced Flk-1/KDR activation. Herbimycin A and PP2,
specific inhibitors for Src family kinases, both inhibited LPC-induced Flk-1/KDR activation in a
concentration-dependent manner. Transfection of KI Src also inhibited LPC-induced Flk-1/KDR
activation. From these findings, it was suggested that c-Src is involved in Flk-1/KDR
transactivation in HUVEC. Moreover, LPC-induced ERK1/2 and Akt activation, which are both
reported to be downstream of Flk-1/KDR (17), were also inhibited by SU1498, VTKi,
Herbimycin A, PP2 and transfection with KI Src. It was also found that LPC-induced Flk-
1/KDR transactivation resulted in a stimulation of HUVEC proliferation. VEGF receptor
inhibitors, MEK1/2 inhibitors, PI3K inhibitors, Src family inhibitors and transfection with KI Src
all inhibited LPC-induced HUVEC proliferation.
The transactivation of RTK in response to the stimulation of a number of GPCRs, such as
transactivation of the EGF receptor by GPCR ligands such as thrombin, angiotensin II,
lysophosphatidic acid, and endothelin-1 has been reported (18, 26). However, LPC-stimulated
transactivation of RTK has not yet been reported or elucidated. Nevertheless the role of LPC in
the pathogenesis of atherosclerosis and systemic autoimmune diseases is well documented (27,
28), even though its specific cell surface receptor was not identified until quite recently. To date,
a subfamily of GPCRs consisting of G2A and GPR4 have been identified as the receptors for
LPC (9, 10). It is reported that G2A is expressed in atherosclerotic lesions (29). On the other
hand, it is reported that G2A and GPR4 are proton-sensing GPCRs (30, 31). However, it is also
reported that GPR119 is a newly identified receptor for LPC (11). Although it still remains to be
13

Page 14

clarified what GPCRs are responsible for LPC action, we found that LPC caused Flk-1/KDR
transactivation in HUVEC. LPC has been implicated in the pathological states of vascular EC
that may be related to the progressive pathological events of atherosclerosis. It has been reported
that the plasma concentration of LPC is elevated in patients with coronary artery diseases
compared to that of normal subjects (32). However, the precise intracellular mechanisms
mediated by LPC stimulation in EC have not yet been clarified. In the present study, we found
for the first time that LPC induces Flk-1/KDR transactivation in HUVEC (Fig.1, 2). Because
Flk-1/KDR activation is reported to be an important factor for atherogenesis (33, 34), LPC-
induced Flk-1/KDR transactivation is taken to be one of the pathological development of
atherosclerosis.
Several mechanisms for RTK transactivation have been proposed. For example, Pyk2,
reactive oxygen species, intracellular free Ca2+, and matrix metalloproteases have been identified
as factors which activate RTK in many types of cells (17, 18). We and others previously
reported that c-Src is involved in the downstream signaling events after stimulation of GPCR and
RTK (15, 16, 19). Therefore, we examined whether c-Src mediates LPC-induced Flk-1/KDR
transactivation in the present study. As shown in Fig. 3, c-Src was rapidly and significantly
activated by LPC in HUVEC. Furthermore, Herbimycin A, PP2 and transfection of KI Src
significantly inhibited LPC-induced Flk-1/KDR transactivation (Fig. 4, 5). From these findings,
it is strongly suggested that c-Src is involved in LPC-induced Flk-1/KDR transactivation and c-
Src may be localized near the receptor and plasma membrane. However, because we did not
determine whether c-Src directly regulates Flk-1/KDR activity in this study, and further studies
are required to define the precise role and the mechanisms of Src kinase in Flk-1/KDR
transactivation.
14

Page 15

One previous report suggested that a PI3K-Akt-eNOS pathway exists downstream of the
Flk-1/KDR transactivation by sphingosine 1-phosphate (S1P), because the S1P-induced Akt and
eNOS activation were inhibited by SU1498 and VTKi (17). In the present study, we found
similarly that LPC-induced ERK1/2 and Akt activation were inhibited by SU1498 and VTKi
(Fig. 6). It is reported that ERK1/2 and Akt are activated by VEGF as early as 5 to 10 min after
administration (35). In the present study, we found that LPC-induced ERK1/2 and Akt
activation were observed. Both the ERK1/2 and Akt activation after transactivation are likely to
have an important role in EC function.
The proliferation of endothelial cells is a prominent feature of atherosclerotic lesions
because it is an important step towards angiogenesis, as well as contributing to the intimal
thickening which develops after endothelial injury (8, 36). LPC is closely related to
atherosclerosis because LPC is a component of the oxLDL which is widely considered to be
major risk factor for atherosclerosis (3, 4). Therefore, we further examined the effect
HUVEC proliferation. LPC induced HUVEC proliferation in a concentration-dependent manner
with the maximal effect of LPC at 20 ?M (Fig. 8A). This result is consistent with the find
Kuhlmann et al. who reported that 20 ?M LPC induces vascular endothelial proliferation (8).
However, we also observed that LPC at higher concentrations than 25 ?M brought about
HUVEC death rather than proliferation (data not shown). At higher concentration than 25 ?M,
LPC has been shown to cause vascular smooth muscle cell apoptosis (37). It is also reported that
LPC can induce GPCR-mediated apoptosis (38). However, in contrast with these findings, it is
reported that LPC at 10 to 20 ?M facilitates cell growth, differentiation and proliferation (8, 39).
In agreement with these later findings, our results show that 20 ?M LPC significantly induced
HUVEC proliferation (Fig. 8A). As the results of pretreatment with SU1498, VTKi, PD98059,
a
of LPC on
ings of
15