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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:18-26

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:18.)
© 2000 American Heart Association, Inc.

Vascular Biology

Hyperexpression and Activation of Extracellular Signal–Regulated Kinases (ERK1/2) in Atherosclerotic Lesions of Cholesterol-Fed Rabbits

Yanhua Hu; Hermann Dietrich; Bernhard Metzler; Georg Wick; Qingbo Xu

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


*    Abstract
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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 {alpha}-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


*    Introduction
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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.


*    Methods
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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 {alpha}-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 {alpha}-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), [{gamma}-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 ({approx}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 {alpha}-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.


*    Results
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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 1Down). 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 2ADown and 2BDown 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 2BDown). 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. {alpha}-Actin–positive SMCs appeared in various stages of lesions, most frequently in advanced lesions (Figure 2DDown). Cells expressing the macrophage antigen identified by the RAM11 antibody24 were observed in all atherosclerotic lesions (Figure 2EDown), 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 2FDown).



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  		Figure 1. Serum cholesterol levels in rabbits. The values of serum cholesterol were measured every 2 or 4 weeks by an enzymatic procedure. Serum cholesterol levels in rabbits fed a 0.2% cholesterol diet (shaded bars) were significantly higher than in those (open bars) receiving the chow diet.



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  		Figure 2. Cell compositions in atherosclerotic lesions. Rabbits receiving a chow or cholesterol-enriched diet for 16 weeks were killed, and aortic tissue fragments were frozen in liquid nitrogen or fixed in 4% buffered (pH 7.2) formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (A and B). Cryostat sections from aortic segments of rabbits fed a 0.2% cholesterol diet for 16 weeks (C through F) were labeled with normal mouse Ig as a negative control (C) or monoclonal antibodies against {alpha}-actin (D), macrophages (E; RAM11), or T lymphocytes (F; L11/135) and visualized with the peroxidase system. Note the presence of positive staining (dark) in lesions. Arrows indicate internal elastic lamina; arrowheads point to examples of positive-stained cells. Magnification x250.

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 3aDown). Nonspecific reactivity was minimal in the negative control labeled with normal mouse serum (Figure 3bDown), whereas the lesion-covered areas in intima from rabbits receiving a cholesterol-rich diet showed increased immunostaining intensity (Figure 3cDown). 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 3cDown).



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  		Figure 3. Immunofluorescence labeling of ERK1/2 in atherosclerotic lesions. Cryostat sections from aortic tissues of rabbits that received normal (a) or cholesterol-enriched (b and c) diet were fixed with cold 5% acetone/methanol for 30 minutes, air-dried, incubated with normal mouse serum (b) or a mouse monoclonal antibody against ERK1/2 (a and c) for 30 minutes at room temperature, and visualized by anti-mouse Ig-TRITC–conjugated rabbit Ig. Open arrow indicates surface of endothelium; solid arrows indicate lesions. Magnification x250.

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 2Up). Therefore, we performed immunofluorescence double staining to identify the cells expressing activated ERK1/2 in lesions. Figure 4aDown through 4c shows data representing double staining with antibodies against phosphorylated ERK1/2 (a; green), {alpha}-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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  		Figure 4. Immunofluorescence double labeling of phosphorylated-ERK1/2 and SMCs in atherosclerotic lesions. Cryostat sections from rabbit aortic tissues 16 weeks after administration of cholesterol-rich diet were incubated with a mouse monoclonal antibody against phosphorylated ERK1/2 conjugated with FITC (a) for 30 minutes. After a washing, sections were incubated with a monoclonal antibody against {alpha}-actin conjugated with Cy3 (b) and stained with Hoechst 33258 (1 µg/mL) for 3 minutes (c). Small arrows indicate internal elastic lamina; large arrows point to examples of double positive–stained cells. Magnification x250.

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 5Down shows that the most PCNA-positive cells had higher levels of phosphorylated ERK1/2.



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  		Figure 5. Immunofluorescence double labeling of phosphorylated ERK1/2 and PCNA in atherosclerotic lesions. Cryostat sections from rabbit aortic tissues 16 weeks after administration of cholesterol-rich diet were incubated with a mouse monoclonal antibody against phosphorylated ERK1/2 conjugated with FITC (a) for 30 minutes. After a washing, sections were incubated with a biotin-labeled monoclonal antibody against PCNA (b), visualized with streptavidin-TRITC, and stained with Hoechst 33258 (1 µg/mL) for 3 minutes (c). Small arrows indicate internal elastic lamina; large arrows point to examples of double positive–stained cells. Magnification x250.

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 6ADown). ERK proteins in intima with lesions were significantly higher than intima/media of control animals and media of cholesterol-fed rabbits (Figure 6ADown, bottom) when they were normalized with respect to actin levels of the same blots.



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  		Figure 6. Elevated ERK proteins and activities in atherosclerotic lesions. Animals were killed 16 weeks after receiving a chow or cholesterol diet, and aortas were harvested. Intima and media (I/M) from chow-fed rabbits and the atherosclerotic intima (AS) and media (M) with lesions from cholesterol-fed rabbits were dissected from the remaining adventitia on ice with tweezers and scissors. Tissues were frozen in liquid nitrogen and homogenized in a Polytron homogenizer. Protein extracts (50 µg/lane) were separated on 10% SDS-polyacrylamide gel, transferred onto membrane, and probed with the antibody against ERK1/2 (A, top) or antibody to phosphorylated-ERK1/2 (P-ERK; B). Immunocomplexes were visualized by a Western blot detection kit. To label ß-actin, the blots were stripped and restained with a monoclonal antibody to actin and developed with the detection kit (A, middle). For the kinase assay, ERK2 proteins were immunoprecipitated from the protein extractions, and their kinase activities (C) were measured on the basis of phosphorylation of MBP substrate. Each lane represents an individual. A, Bottom, Graph of pan-ERK1/2 (mean±SD) obtained from 6 rabbits per group, which were normalized with respect to actin of corresponding blots by measurement of optical densities. *Significant difference from control, P<0.05.

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 6BUp). 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 6CUp 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 6CUp).

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 7Down 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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  		Figure 7. Percentage of SMC outgrowth in cultured explant arterial fragments. The intima with lesions from cholesterol-fed rabbits and the intima and inner half of media from chow-fed rabbits were carefully dissected from the vessel, cut into pieces ({approx}1 mm3), explanted onto a gelatin-coated plastic bottle, and incubated at 37°C in medium. The outgrowths of SMCs from the explants were counted at days 5, 10, and 15 under the microscope. Data are mean±SD from 3 independent experiments. *Significant difference from controls, P<0.05.

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 8ADown). 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 8BDown). 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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  		Figure 8. Enhanced ERK activation in lesional SMCs. A, Lesional SMCs were serum-starved for 2 days, exposed to normocholesterolemic (n-LDL; 200 µg/mL) or hypercholesterolemic (Chol-LDL; 200 µg/mL) LDL for 10 minutes, and harvested for protein extracts. B, Both normal and lesional SMCs were serum-starved for 2 days; stimulated with hypercholesterolemic LDL (200 µg/mL), PDGF-AB (100 ng/mL), or serum (10% FCS) for 10 minutes; and harvested for protein extracts. For the kinase assay, ERK2 proteins were immunoprecipitated from the protein extractions, and their kinase activities were measured on the basis of phosphorylation of MBP substrate. Graph in B shows ERK2 kinase activities (mean±SD) of lesional SMCs normalized to normal SMCs (zero in y-axis indicates 100%). S indicates FCS treatment as a positive control; Ctl, negative controls.

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 9ADown); 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 9BDown 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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  		Figure 9. PD 98059 inhibited ERK activation and SMC proliferation. A, Quiescent SMCs were pretreated with PD 98059 for 30 minutes. Aortic SMCs derived from normal rabbits were exposed to LDL for 10 minutes and harvested for protein extracts. The results of Western blot analysis showed LDL-induced (200 µg/mL) ERK1/2 phosphorylation (P-ERK) inhibited by PD 98059 in a concentration-dependent manner. B, For proliferation assays, SMCs (1x104) cultured in a 96-well plate in medium containing 10% FCS at 37°C for 24 hours were serum-starved for 2 days and treated with PD 98059, and LDL (200 µg/mL) in 2% serum was added and incubated at 37°C for 24 hours. [3H]thymidine was added 6 hours before cells were harvested. Radiation activities were measured. Data are mean±SD of 3 experiments.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 3Up and 6Up) 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.


*    Acknowledgments
 
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.


*    References
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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