© 2000 American Heart Association, Inc.
Vascular Biology |
Hyperexpression and Activation of Extracellular Signal–Regulated Kinases (ERK1/2) in Atherosclerotic Lesions of Cholesterol-Fed Rabbits
From the Institute for General and Experimental Pathology (Y.H., H.D., G.W.) and Division of Cardiology, Department of Internal Medicine (B.M.), University of Innsbruck Medical School, and the Institute for Biomedical Aging Research (G.W., Q.X.), Austrian Academy of Sciences, Innsbruck, Austria.
Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail qingbo.xu{at}oeaw.ac.at
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Abstract |
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Abstract—A hallmark of hyperlipidemia-induced atherosclerosis is altered gene expression that initiates cell proliferation and (de)differentiation in the intima of the arterial wall. The molecular signaling that mediates this process in vivo has yet to be identified. Extracellular signal–regulated kinases (ERKs) are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate the activity, abundance, and localization of ERK1/2 in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of ERK1/2, mainly localized in the cap and basal regions of atheromas. A population of ERK-enriched cells was identified as
-actin–positive
smooth muscle cells (SMCs). ERK1 and 2 were heavily
phosphorylated on tyrosyl residues and coexpressed with
proliferating cell nuclear antigen in atherosclerotic lesions. ERK1/2
protein levels in protein extracts from atherosclerotic lesions were 2-
to 3-fold higher than the vessels of chow-fed rabbits, and their
activities were elevated 3- to 5-fold over those of the normal vessel.
SMCs derived from atherosclerotic lesions had increased
migratory/proliferative ability and higher ERK activity in response to
LDL stimulation compared with cells from the normal vessel. Inhibition
of ERK activation by PD98059, a specific inhibitor of
mitogen-activated protein kinase kinases (MEK1/2), abrogated
LDL-induced SMC proliferation in vitro. Taken together, our findings
support the proposition that persistent activation and hyperexpression
of ERK1/2 may be a critical element to initiate and perpetuate cell
proliferation during the development of atherosclerosis.
Key Words: atherosclerosis • animal models • MAP kinases • ERK • signal transduction
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Introduction |
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Mitogen-activated protein (MAP) kinase–mediated signal transduction pathways contribute to cell growth and differentiation.1 2 3 The MAP kinase isoforms of 42 and 44 kDa, so-called extracellular signal–regulated kinases (ERK1/2), are expressed in most, if not all, mammalian cell types. ERK1 and 2 were initially identified as 2 protein kinases that became phosphorylated on tyrosine in response to growth factors.4 ERK-mediated signal pathways are a multistep phosphorylation cascade that transmits signals from the cell surface to cytosolic nuclear targets, which are responsible for the activation and phosphorylation of a number of other regulatory proteins, including p90rsk, cPLA2, and transcription factors needed for the expression of genes involved in cell proliferation.5 6 7 In addition, the activation of the cascade is also required for passing through certain checkpoints in the cell cycle, eg, G1/S and G2/M, in proliferating cells in vitro.8 9 10 Therefore, MAP kinase–mediated signal pathways play a key role in initiating cell proliferation and differentiation.
A high concentration of circulating cholesterol or LDLs is believed to be a major risk factor for atherosclerosis. The main pathophysiological role of LDL is to deliver cholesterol to vascular smooth muscle cells (SMCs) and macrophages, which form foam cells in the development of atherosclerosis.11 In addition to lipid transport, LDL can effectively stimulate SMC proliferation, a key event in the formation of atherosclerosis.12 13 14 There is evidence that LDL induces gene expression of platelet-derived growth factor (PDGF), PDGF receptors, c-fos, and egr-1,14 15 which are essential transcription factors for SMC proliferation. However, the precise signal transduction pathways that link to hypercholesterolemia and quantitative changes in gene expression in the pathogenesis of atherosclerotic lesions are largely unknown.
Most of our knowledge concerning the activation and function of ERK1/2 has come from studies on cultured cells; little is known about their activation in vivo and their relevance to atherogenesis in animal models. We examined ERK1/2 expression, localization, and activation in atherosclerotic lesions of cholesterol-fed rabbits and provide the first evidence of ERK overexpression and activation in lesions. Moreover, we demonstrate that the increased migratory/proliferative ability of SMCs derived from the lesions correlates with ERK1/2 activities, which are induced by LDL from chow- and cholesterol-fed rabbits.
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Methods |
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Rabbit Model for Atherosclerosis
Twenty New Zealand White male rabbits weighing between 1800 and 2200 g were obtained from Charles River (Kisslegg, Germany). All animals were selected for serum cholesterol levels <100 mg/dL and were individually housed in wire-bottomed cages at 22°C with a relative humidity of 55%. All received water ad libitum and were fed either a normal standard chow diet (T775; Tagger & Co) or a cholesterol-enriched diet (0.2% wt/wt) for 16 weeks, as described previously.16 17 Animals were killed by heart puncture under ketamine (25 mg/kg) and xylazine (5 to 10 mg/kg) anesthesia. Serum was collected for cholesterol assays and LDL isolation. The aortas were carefully removed intact from the aortic arch to the iliac bifurcation, immediately placed into cold PBS (4°C), and prepared for histological analysis, tissue culture, and protein extractions. For conventional histology, tissue fragments were fixed in 4% buffered (pH 7.2) formaldehyde, embedded in paraffin, and sectioned for hematoxylin-eosin staining.
Blood Cholesterol
Blood (1 to 2 mL) was taken from the central ear artery of
rabbits that had been fasted for 16 hours. Serum total
cholesterol values were measured every 2 or 4 weeks by an
enzymatic procedure (Sigma). Briefly, 10 µL serum was added to 1 mL
solution of cholesterol test kit and incubated for 18
minutes at room temperature followed by photometer measurement at
500-nm excitation wavelength (Dynatech Laboratories Inc).
Immunohistochemical and Immunofluorescence
Double Staining
The procedure used for immunohistochemical staining was similar
to that described elsewhere.16 17 Briefly, serial
4-µm-thick frozen sections were overlaid with mouse monoclonal
antibodies against -actin (Sigma), macrophages (RAM11;
Dako), or CD3+ T cells (L11/135; ATCC; catalogue
No. TIB188); incubated with rabbit anti-mouse Ig conjugated with
peroxidase (Dako); and developed for 20 minutes at room temperature
with a substrate solution.
For immunofluorescent staining, a mouse monoclonal antibody
against ERK1/2 (Transduction) was added to the sections. After 3 washes
with PBS, the sections were incubated with a rabbit anti-mouse
Ig–TRITC conjugate (Dako) for 30 minutes. For double staining,
sections were incubated with a monoclonal antibody against
phosphorylated ERK1/2 conjugated with FITC (Santa Cruz
Biotechnology Inc), rinsed, and stained with a mouse monoclonal
antibody against -actin–Cy3 conjugate (Sigma) or biotin-labeled
antibody against proliferating cell nuclear antigen (PCNA) developed
with streptavidin-TRITC (Dako). For visualization of nuclei, sections
were counterstained with the DNA stain Hoechst 33258 (1 µg/mL PBS;
Lambda Probes) for 1 minute. Sections were mounted in
Gelvatol/PBS and examined in a epi-illumination
immunofluorescence microscope equipped with
appropriate filter combinations for the 3-wavelength method
(Leitz).
Protein Extraction
The procedure used for protein extracts was similar to that
described previously,18 with a slight modification.
Briefly, the atherosclerotic intima and media with lesions were
dissected from the remaining adventitia on ice with tweezers and
scissors. Tissues were frozen in liquid nitrogen and
homogenized in a Polytron homogenizer in
buffer A containing 20 mmol/L HEPES (pH 7.4), 50 mmol/L
ß-glycerophosphate, 2 mmol/L EGTA, 1 mmol/L DTT, 1
mmol/L Na3VO4, 1% Triton
X-100, 10% glycerol, 1 µg/mL leupeptin, 400 µmol/L PMSF, and
1 µg/mL aprotinin. The homogenate was incubated on ice
for 15 minutes. After centrifugation at
17 000g for 30 minutes, the supernatant was collected, and
protein concentration was measured with Bio-Rad protein assay
reagent.
Western Blot Analysis
Protein extracts (50 µg/lane) prepared from the
arterial tissues described above were separated by
electrophoresis through a 10% SDS-polyacrylamide gel and
transferred to an Immobilon-P transfer membrane.19 The
membranes were processed with the monoclonal antibody against ERK1/2 or
phosphorylated ERK1/2 (Santa Cruz
Biotechnology). Specific antigen-antibody complexes were then
detected with the ECL Western Blot Detection Kit (Amersham). The blots
were stripped for 30 minutes at 70°C in the buffer containing 60
mmol/L Tris, 2% SDS, and 100 mmol/L 2-mercaptoethanol; labeled
with a monoclonal antibody against ß-actin (Sigma); and developed as
described above. Graphs of blots were obtained in the linear range of
detection and were quantified and normalized to the level of actin by
scanning laser densitometry (Power-Look II, UMAX Data System Inc) of
graphs.
Kinase Assay
Supernatant (0.5 mL) containing 0.5 mg proteins was incubated
with 10 µL of goat anti-ERK2 antibodies (Santa Cruz Biotechnology)
for 2 hours at 4°C with rotation. Subsequently, 40 µL of protein
G–agarose suspension (Santa Cruz Biotechnology) was added, and
rotation continued for 1 hour at 4°C. The immunocomplexes were
precipitated by centrifugation and washed 2 times with
buffers A, B (500 mmol/L LiCl, 100 mmol/L Tris, 1 mmol/L
DTT, 0.1% Triton X-100; pH 7.6), and C (20 mmol/L MOPS, 2
mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L
DTT, 0.1% Triton X-100; pH 7.2), respectively. ERK2 activities in the
immunocomplexes were measured as described
previously.18 19 Briefly, immunocomplexes were incubated
with 35 µL of buffer C supplemented with myelin basic protein (MBP; 6
µg; Upstate Biotechnology), [
-32P]ATP (5
µCi), and MgCl2 (50 mmol/L) for 20 minutes
at 37°C, with vortexing every 5 minutes. To stop the reaction, 15
µL of 4x Laemmli buffer was added, and the mixture was boiled for 5
minutes. Proteins in the kinase reaction were resolved by SDS-PAGE
(15% gel) and subjected to autoradiography.
Cell Culture and Proliferation Assays
Rabbit vascular SMCs were cultivated from their aortas by a
modification of the procedure described by Ross and
Kariya.20 21 In short, thoracic aortas of chow- and
cholesterol-fed rabbits were removed and washed with RPMI
1640 medium (Gibco). Intima with lesions and normal media were
carefully dissected from the vessel, cut into pieces (
1
mm3), and explanted onto a 0.02% gelatin-coated
plastic bottle (Falcon). The bottle was incubated upside-down at 37°C
in a humidified atmosphere of 95% air/5% CO2
for 3 hours, resulting in firm attachment of the explanted tissues, and
then medium supplemented with 20% FCS, penicillin (100 U/mL), and
streptomycin (100 µg/mL) was slowly added. The outgrowths of SMCs
from the explants were counted at days 5, 10, and 15 under the
microscope. The percentage of the outgrowth was determined by counting
positive tissue explants with cell growth over all explanted tissue
segments (50 to 100 pieces per bottle). Cells were passaged by
treatment with 0.05% trypsin/0.02% EDTA solution. Experiments were
conducted on SMCs between passages 5 and 10 that had just achieved
confluence. The purity of SMCs was routinely confirmed by
immunostaining with antibodies against -actin.
For proliferation assays, SMCs (1x104) cultured in 96-well plates in medium containing 10% FCS at 37°C for 24 hours were serum-starved for 2 days. SMCs were treated with PD 98059 (Calbiochem) for 30 minutes, and then LDL (100 µg/mL) in 2% serum was added and incubated at 37°C for 24 hours. [3H]thymidine was added 6 hours before cell harvest. Radiation activities were measured.
LDL Isolation
EDTA plasma was collected from
normocholesterolemic and
hypercholesterolemic rabbits fasted overnight.
Lipoproteins were prepared by differential
centrifugation with solid KBr to adjust the density as
described previously.22 23 LDLs were obtained in the
fraction between 1.020 and 1.050 g/mL. The sample was dialyzed against
150 mmol/L NaCl with 0.1 mmol/L EDTA, sterilized through a
0.2-µm Millipore membrane, and stored at 4°C up to 3 weeks. No
oxidation of LDL was observed at least 3 weeks after LDL isolation, as
determined by measurement of malondialdehyde by the thiobarbituric acid
method. Endotoxin contents of freshly isolated LDL and LDL after 3
weeks of storage at 4°C were both below the detection limit (<1
ng/mL, Endotoxin Kit, Sigma). Concentrations of LDL were determined
gravimetrically by aliquot weight after drying, and quantities of
lipoproteins were expressed as total weights.22 23
Statistical Analysis
ANOVA was performed for multiple comparisons. The Mann-Whitney
U test was used for comparison between 2 groups. A value of
P<0.05 was considered statistically significant.
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Results |
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Atherosclerotic Lesions in Hypercholesterolemic Rabbits
Blood cholesterol levels in rabbits receiving a 0.2% cholesterol diet were significantly elevated and reached 350 mg/dL at 2 weeks and >600 mg/dL at 16 weeks. Animals in the control group (chow diet) had blood cholesterol levels <100 mg/dL (Figure 1
). To verify
atherosclerotic lesions in cholesterol-fed rabbits, aortas
were examined morphologically and immunohistologically
16 weeks after cholesterol feeding. Areas with lesions in
the surface of aortic intima covered 50% to 80% of intima in
cholesterol-fed rabbits. Figure 2A and 2B depicts the
histological appearance of vessel walls of rabbits that
received chow and cholesterol diets, respectively. Intima
of normal vessel walls constituted a monolayer
endothelium and a little connective tissue, but aortic
lesions of rabbits fed a cholesterol-enriched diet were
characterized by cell proliferation, foam cell accumulation, and lipid
deposition in the intima (Figure 2B). To identify the main
cellular components in atherosclerotic lesions, serial sections of the
vessels were incubated with a battery of antibodies to specific cell
markers. -Actin–positive SMCs appeared in various stages of
lesions, most frequently in advanced lesions (Figure 2D). Cells
expressing the macrophage antigen identified by the RAM11
antibody24 were observed in all atherosclerotic lesions
(Figure 2E), including early and advanced stages, most appearing
as foam cells. Finally, T lymphocytes identified by monoclonal antibody
L11/135 were frequently observed, especially in advanced lesions
(Figure 2F).
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ERK Hyperexpression and Activation in Atherosclerotic
Lesions
From each group, 5 aortic specimens were
immunohistologically stained with a monoclonal antibody
against mammalian ERK1/2. Normal artery showed very weak staining, if
any (Figure 3a). Nonspecific reactivity
was minimal in the negative control labeled with normal mouse serum
(Figure 3b), whereas the lesion-covered areas in intima from
rabbits receiving a cholesterol-rich diet showed increased
immunostaining intensity (Figure 3c). In the
small lesions, some areas within the intima became positively stained,
whereas fatty streaks displayed elevated ERK1/2 content in
subendothelial regions. Heterogeneity
of ERK1/2 staining became more evident in atherosclerotic plaques.
Sites of increased ERK1/2 were mainly within the cap and base regions
of the atherosclerotic plaque (Figure 3c).
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Both ERK1 and ERK2 kinases are activated by dual
phosphorylation of tyrosine and threonine residues in
response to mitogenic or stress stimuli.1 4 In
addition, tissues derived from atherosclerotic lesions are
heterogeneous with respect to cell types (Figure 2
).
Therefore, we performed immunofluorescence double
staining to identify the cells expressing activated ERK1/2 in
lesions. Figure 4a through 4c shows data
representing double staining with antibodies against
phosphorylated ERK1/2 (a; green), -actin (b; red),
and counterstaining with Hoechst 33258 (c; blue). Typical
double-positive cells are indicated by arrows, demonstrating a
population of SMCs in lesions expressing activated ERK1/2. In
addition, some macrophages were also positively stained with
phosphorylated ERK1/2, indicating an activated
or proliferating state.
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There is evidence that ERK activation is required for passing through
certain checkpoints in the cell cycle in proliferating cells in
vitro.8 9 10 We performed experiments with
immunofluorescence double staining with antibodies
against phosphorylated ERK1/2 (a; green), PCNA (b;
red), and counterstaining with Hoechst 33258 (c; blue). Figure 5 shows that the most PCNA-positive cells
had higher levels of phosphorylated ERK1/2.
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To further show that ERK1/2 proteins were increased in
atherosclerotic lesions, protein extracts from tissues of normal
intima/media and intima and media with lesions were analyzed by
Western blot analysis. Abundant ERK1/2 proteins in
atherosclerotic lesions were observed (Figure 6A). ERK proteins in intima with lesions
were significantly higher than intima/media of control animals and
media of cholesterol-fed rabbits (Figure 6A, bottom)
when they were normalized with respect to actin levels of the same
blots.
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Western blot analysis using protein extracts from the
arterial tissues and the antibody recognizing the
phosphorylated ERK1/2 was also performed. The
activated (phosphorylated) forms of p42 and p44
were identified, which showed marked increases in protein extracts of
atherosclerotic lesion tissues (Figure 6B). These results
demonstrated that ERK phosphorylation is present in
lesions. Furthermore, ERK1/2 activity of protein extracts was also
measured with MBP used as a substrate. Figure 6C shows the
results of an experiment examining ERK1/2 activities in the vessel
wall. Obviously, ERK1/2 activities were found at low levels in control
vessels and media of cholesterol-fed rabbits but increased
3- to 5-fold in atherosclerotic lesions (Figure 6C).
Increased ERK Activities in Lesion-Derived SMCs
To compare cell proliferation and ERK activation between SMCs from
atherosclerotic lesions and normal vessels, tissues were explanted onto
gelatin-coated bottles, and SMC migration and/or proliferation from the
tissues was evaluated microscopically. Data shown in Figure 7 are percentages of outgrowth SMCs
around the tissue fragments. The results indicate that the
migration/proliferation ability of SMCs from lesions was significantly
higher than that of normal vessels.
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To compare ERK activities in different types of SMCs, cellular protein
extracts containing similar amounts of actin were used for
immunoprecipitation with the specific antibody against ERK2, and kinase
activities were measured on the basis of
phosphorylation of basic myelin protein as a substrate.
When lesion-derived SMCs were stimulated with LDL of
normocholesterolemic or
hypercholesterolemic rabbits, ERK2 activation was
induced by both types of LDL at similar magnitudes (Figure 8A). However, ERK2 activities in
lesion-derived SMCs stimulated with
hypercholesterolemic LDL, PDGF, and serum were higher
than those of SMCs derived from normocholesterolemic
rabbits (Figure 8B). Taken together, these observations support
the notion that alterations in ERK activation in the development of
atherosclerosis of cholesterol-fed rabbits
are due to changed sensitivity of SMCs to LDL and mitogen
stimulation.
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Inhibition of ERK Activation and SMC Proliferation
Because ERK-mediated signal pathways are crucial in mediating cell
migration and proliferation, the effects of PD98059, a MAP kinase
kinases 1/2 inhibitor, on LDL-stimulated ERK
activation and SMC proliferation were investigated. A marked activation
of ERK1/2 by LDL was found, which was inhibited by PD98059 in a
concentration-dependent manner (Figure 9A); ERK2 kinase
phosphorylation was completely abolished by 50
µmol/L PD98059. We had observed, by measuring
[3H]thymidine incorporation, that LDL
effectively induced SMC DNA synthesis. Figure 9B depicts PD98059
inhibition of SMC proliferation stimulated by LDL. A concentration of
50 µmol/L for treatment of SMCs completely abrogated SMC
proliferation. Thus, blocking ERK-mediated signaling inhibits SMC
proliferation induced by LDL.
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Discussion |
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Activation of ERK kinase cascades is one of the major pathways for the regulation of proliferation and cell growth in various cultured cells.1 2 3 4 25 26 Reversible protein phosphorylation is the established mechanism of regulation of ERK kinases,27 an activity controlled by a family of dual-specificity protein kinases and a complex upstream cascade.4 A transient kinase activation or attenuation is seen in most in vitro cultured cells in response to chronic stimulation. In the present studies, we have demonstrated that in vivo, ERK activation is sharply elevated in lesions, which cannot be ascribed solely to phosphorylation of the proteins. Immunoblotting revealed a marked increase in the amount of ERK1/2 proteins from atherosclerotic lesions compared with normal vessel tissues or aortic media of cholesterol-fed rabbits. Sustained expression and activation of ERK kinases in ERK-transfected fibroblasts resulted in oncogenicity associated with cell proliferation.28 Hyperexpression and activation of these kinases may play a main role in regulation of cell proliferation in the pathogenesis of atherosclerosis.
Chamley-Campbell et al29 hypothesized that 2 distinct SMC phenotypes, contractile and synthetic, exist in the vessel wall and that SMCs in the atherosclerotic plaque differ from those in the normal tunica media.30 It has been established that SMCs in intimal lesions display increased levels of genes for growth factors,14 tumor necrosis factors,31 class II histocompatibility antigens,32 vascular cell adhesion molecule-1,33 and intercellular adhesion molecule-1.34 35 On the basis of these observations, Libby and Li36 called them "activated" SMCs. Our findings of selective or differential hyperexpression and activation of ERK protein kinases in atherosclerotic SMCs support the concept that the ERK level and activity in SMCs reflect a situation of gene expression, activation, and replication of this SMC population. These higher ERK activities from lesional SMCs can be maintained even in in vitro culture for longer periods of time, further supporting the notion that SMCs from atherosclerotic lesions have been selected and differ from those from normal vessels.
Proliferation of vascular SMCs is a hallmark in the pathogenesis of
atherosclerosis.14 LDL and oxidized LDL
are mitogenic to cultured SMCs and have been demonstrated
to activate ERK signal pathways in cultured cells in
vitro.37 38 39 40 In the present study, we provide the
first evidence that hypercholesterolemia can
stimulate ERK expression and activation in the intima but not media or
tissues (Figures 3 and 6) from other organs, including
liver, kidney, brain, and heart (data not shown). Previously, we
demonstrated that acute elevation in blood pressure induced by
restraint or hypertensive agents resulted in MAP kinase activation in
the media of the arterial wall.41 In the
present experiments, we minimized the effect of animal handling on
blood pressure fluctuation by daily conditioning of rabbits with
intramuscular saline injection for 1 week before their death. This
treatment was shown to be effective because kinase activities of the
vessel wall from control rabbits and media from
cholesterol-fed rabbits were of similar, and lower, levels.
Such tissue-specific activation of ERK kinases induced by
hypercholesterolemia may explain why the lesion
is localized only in the arterial intima and may be due to
different responses of various types of cells to lipids or LDL
stimulation, ie, hypercholesterolemia induces
atherosclerosis but not kidney or heart sclerosis.
Thus, our findings could significantly enhance our understanding of the
pathogenesis of atherosclerosis during
hyperlipidemia.
Recent studies have focused on the signaling events in cultured cells from cardiovascular tissue, including myocytes and SMCs, which may provide a new strategy for therapeutic intervention.3 25 42 43 Depletion of MAP kinases with an antisense oligodeoxynucleotide downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes.44 Accumulating evidence indicates that MAP kinase phosphatase (MKP-1) specifically inhibits mitogen-induced activation of MAP kinases in cell lines.45 46 47 Lai et al48 reported a reduction of MKP-1 expression in rat carotid arteries in response to balloon injury, which may be responsible for sustained activation of ERK2 during restenosis of the injured artery.49 In the present study, we demonstrate that inhibition of the ERK kinase activation by PD98059 abrogates SMC proliferation. The therapeutic effect of ERK antagonist or inhibitor on lesion formation should be addressed in future studies. Thus, understanding of the mechanisms serving to regulate MAP kinase activities could lead to new strategies for prevention or therapeutic intervention for atherosclerosis.
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Acknowledgments |
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This work was supported by grants P-12568-MED and P-13099-BIO (to Q. Xu) from the Austrian Science Fund and 6286 (to Q. Xu) from the Jubiläumsfonds of the Austrian National Bank. Dr Hu is a recipient of an APART Stipend from the Austrian Academy of Sciences. We thank A. Jenewein, E. Rainer, and G. Sturm for excellent technical assistance and T. Öttl for the preparation of photographs.
Received February 26, 1999; accepted July 28, 1999.
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