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From the Division of Cardiology,*
Department of Internal Medicine,
and the Institute for General and Experimental
Pathology,
University of Innsbruck Medical
School; and the Institute for Biomedical Aging
Research,
Austrian Academy of Sciences,
Innsbruck, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin+ SMCs and
RAM11+ macrophages contained abundant phosphorylated
SAPK/JNK proteins. SAPK/JNK protein levels in protein extracts from
atherosclerotic lesions were two- to threefold higher than the vessels
of chow-fed rabbits. SAPK/JNK activities were elevated three- to
fivefold higher than the normal vessels. Interestingly,
increased SAPK/JNK in lesions was co-localized or coincided with high
levels of transcription factor p53 as identified by double labeling and
immunoprecipitation. Abundant pro-apoptotic protein BAX and
BCL-XS were also observed. Furthermore, low-density
lipoprotein (LDL) and oxidized LDL stimulated SAPK/JNK activation in
cultured SMCs in a time- and dose-dependent manner. LDL also induced
SAPK/JNK activation in vascular SMCs derived from
LDL-receptor-deficient Watanabe rabbits, indicating a
LDL-receptor-independent process. Thus, SAPK/JNK persistently
hyperexpressed and activated in lesions may play a key role in
mediating cell differentiation and apoptosis during the development of
atherosclerosis via activation of transcription factor
p53.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The atherosclerotic lesion is defined by arterial intimal cell proliferation, lipid accumulation, and connective tissue deposition. The major cellular components of the human atherosclerotic plaques are smooth muscle cells (SMCs), which dominate the fibrous cap, and macrophages, which are the most abundant cell type in the lipid-rich core region.24,25 Although the pathogenic mechanism of atherosclerosis is not fully understood, a high concentration of circulating cholesterol or low-density lipoproteins (LDL) is believed to be a major risk factor. LDL delivers cholesterol to vascular SMCs and macrophages, stimulates cell proliferation, and induces gene expression of platelet-derived growth factor (PDGF), PDGF receptors, c-fos, and egr-125,26 in the arterial wall. LDL or oxidized LDL stimulation can also evoke cell apoptosis,27-29 which has been observed in atherosclerotic lesions.30-33 In addition, p53 has been identified to be highly expressed in atherosclerotic lesions and to be involved in oxidized LDL-induced apoptosis.27 However, the precise signal transduction pathways that link hypercholesterolemia or LDL stimulation to quantitative changes in gene expression and, to apoptosis in the pathogenesis of atherosclerosis, remain to be elucidated. In the present study, we examined SAPK/JNK expression, localization, and activation in atherosclerotic lesions of cholesterol-fed rabbits and provide the first evidence of SAPK/JNK overexpression and activation in lesions. Furthermore, we demonstrate that LDL and oxidized LDL stimulate SAPK/JNK activation in cultured SMCs independent of classic LDL receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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New Zealand White male rabbits weighing between 1,800 and 2,200 g were obtained from Charles River (Kissleg, Germany). All animals were selected for serum cholesterol levels under 100 mg/dl, 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., Graz, Austria) or a cholesterol-enriched diet (0.2% w/w) for 16 weeks, as described previously.34,35 Animals were sacrificed 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 put into cold phosphate-buffered saline (PBS) (4°C) and prepared for histological analysis, tissue culture, and protein extractions. The total surface area of aortas covered by lesions was evaluated. For conventional histology, tissue fragments were fixed in 4% buffered (pH 7.2) formaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining. Serum cholesterol values were measured every 2 or 4 weeks using an enzymatic procedure (Sigma Chemical Co., St. Louis, MO).
Immunohistochemical and Immunofluorescence Double Staining
The procedure used for immunohistochemical staining was similar to
that described elsewhere.34,35
Briefly, serial
4-µm-thick frozen sections were overlaid with mouse monoclonal
antibodies against -actin (Sigma), macrophages (RAM11; DAKO,
Copenhagen, Denmark), or T cells (L11/135; ATCC, Rockville, MD) and
incubated with rabbit anti-mouse immunoglobulin (Ig) conjugated with
peroxidase (DAKO) and developed for 20 minutes at room temperature
using a substrate solution.
For immunofluorescence staining, a monoclonal antibody against SAPK/JNK1 (Transduction Lab., Lexington, KY) was added to the sections. After three washes with PBS, the sections were incubated with a rabbit anti-mouse Ig-tetramethylrhodamine B isothiocyanate (TRITC) conjugate (DAKO) for 30 minutes, or sections were incubated with a monoclonal antibody against phosphorylated SAPK/JNK conjugated with fluorescein isothiocyanate (FITC) (p-JNK; Santa Cruz Biochem., Santa Cruz, CA), mounted in glycerol/PBS, and examined in an epiillumination immunofluorescence microscope equipped with appropriate filter combinations for the three wavelength method (Leitz, Wetzlar, Germany).
For immunofluorescence double staining, frozen sections were incubated
with monoclonal antibodies against -actin Cy3 conjugate (Sigma) or
RAM11 macrophages. After three washes with PBS, the sections were
incubated with a rabbit anti-mouse Ig-TRITC conjugate (DAKO) for 30
minutes, rinsed, blocked with normal mouse serum (1:5), and stained
with a mouse monoclonal antibody against phosphorylated-SAPK/JNK-FITC
conjugate (p-JNK). For p53 and JNK1 double staining, a mouse monoclonal
anti-p53 antibody and rabbit anti-JNK1 antibodies (Santa Cruz Biotech.
Inc.) were used and visualized with swine anti-mouse Ig-FITC and goat
anti-rabbit Ig-TRITC. For visualization of nuclei, sections were
counterstained with the DNA stain Hoechst 33258 (1 µg/ml PBS; Lambda
Probes, Graz, Austria) for 1 minute.
Western Blot Analysis
The procedure used for protein extracts was similar to that described previously36 with a slight modification. Briefly, the atherosclerotic-lesioned intima and media were dissected on ice from the remaining adventitia with tweezers and scissors. Tissues were homogenized in buffer A (20 mmol Hepes, pH 7.4, 50 mmol/L ß-glycerophosphate, 2 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 1 µg/ml leupeptin, 100 µmol/L phenylmethyl sulfonyl fluoride (PMSF), and 1 µg/ml aprotinin), and proteins were extracted. Proteins (50 µg/lane) were separated by electrophoresis through a 10% SDS-polyacrylamide gel and transferred to an immobilon-p transfer membrane. The membranes were processed with monoclonal antibodies against SAPK/JNK1, actin, p53, BAX, and BCL-XS/L (Santa Cruz Biotech. Inc.), respectively, and specific antigen-antibody complexes were detected with the ECL Western Blot Detection Kit (Amersham Co., Little Chalfont, UK).
Immunoprecipitation and Kinase Assay
One-half milliliter of the supernatant containing 0.5 mg proteins
was incubated with 10 µl of rabbit anti-SAPK/JNK1 antibodies and 40
µl of protein A-agarose suspension (Santa Cruz Biochem.). The
immunocomplexes were precipitated by centrifugation and washed 2 times
with buffer A, buffer B (500 mmol/L LiCl, 100 mmol/L Tris, 1 mmol/L
dithiothreitol, 0.1% Triton X-100, pH 7.6), and buffer C (20 mmol/L
Mops, 2 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L
dithiothreitol, 0.1% Triton X-100, pH 7.2), respectively. The
activities of SAPK/JNK in the immunocomplexes were measured as
described previously.11,16
Briefly, immunocomplexes were
incubated with 35 µl of buffer C supplemented with glutathione
S-transferase (GST)-c-Jun (5 µg),
-32P-ATP (Amersham, Little Chalfont, UK), and
MgCl2 (50 mmol/L) for 20 minutes at 37°C with
vortexing every 5 minutes. The GST-c-Jun fusion protein (plasmid
provided by Dr. Woodgett, Toronto, Canada) was produced in
Escherichia coli and isolated using glutathione Sepharose 4B
RediPack Columns (Pharmacia Biotech Inc., Piscataway, NJ) according to
the manufacturer’s protocol. 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-polyacrylamide gel
electrophoresis (12% gel) and subjected to autoradiography.
For immunoprecipitation of SAPK/JNK1 and p53 Western blot analysis, 1 ml of the supernatant containing 2 mg of protein was incubated with 50 µl of rabbit anti-SAPK/JNK1 antibodies and 100 µl of protein A-agarose suspension. The immunocomplexes were precipitated as described for the kinase assay and resolved in SDS-polyacrylamide gel electrophoresis (15% gel).
Cell Cultures
Human and rabbit vascular SMCs were cultivated from arteries using the procedure described by Ross and Kariya37 with a slight modification.38 In short, specimens of human carotid arteries were obtained from the Department of Vascular Surgery, University Hospital of Innsbruck. Intima and inner layer of media were dissected from the arteries and cut into pieces, digested with collagenase and elastase, and cultured in Dulbecco’s modified essential medium (DMEM; Life Technologies, Inc., Grand Island, NY). Thoracic aortas of chow- and cholesterol-fed rabbits were removed, and lesioned intima and normal media were carefully dissected from the vessel, cut into pieces (~1 mm3), and implanted onto a gelatin-coated (0.02%) plastic bottle (Becton Dickinson, Oxnard, CA). The bottle was incubated up-side-down at 37°C in a humidified atmosphere of 95% air/5% CO2 for 3 hours, and then medium supplemented with 20% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) was slowly added. Cells were passaged by treatment with 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) solution. Experiments were conducted on SMCs that had just achieved confluence. Similarly, aortic SMCs from Watanabe rabbits (La Roche, Basel, Switzerland) were cultivated and used for SAPK/JNK kinase assays.
LDL Isolation and Oxidation
EDTA plasma was pooled from normolipemic, fasting (12 to 14 hours) male and female donors, aged 20 to 30 years. Lipoproteins were prepared by differential centrifugation using solid KBr to adjust the density, as described previously.39,40 LDL were obtained in fraction between 1.020 to 1.050 g/ml. Concentrations of LDL were determined gravimetrically by aliquot weight after drying, and quantities of lipoproteins were expressed as total weights.39,40 LDL oxidation was performed by incubation of LDL (1 mg/ml PBS) with 10 µmol/L CuCl2 at 37°C for 18 hours.41 The extent of oxidation was assessed by measurement of thiobarbituric acid reactive substances (TBARS) (9.8 ± 1.3 nmol/mg).42
Statistical Analysis
The Mann-Whitney U test was used for comparison between two groups. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals in the control group (chow diet) had blood cholesterol
levels <100 mg/dl, whereas blood cholesterol levels in rabbits
receiving the 0.2% cholesterol diet were significantly elevated and
reached 500 mg/dl at 4 weeks, and ~700 mg/dl at 16 weeks. Rabbit
aortas from both groups were examined morphologically and
immunohistologically 16 weeks after chow or cholesterol feeding. Fifty
percent to 80% of aortic intima was covered by atherosclerotic
lesions, including fatty streaks and plaques in cholesterol-fed
rabbits. Aortic lesions of rabbits fed with a cholesterol-enriched diet
were characterized by cell proliferation, foam cell accumulation, and
lipid deposition in the intima. Atherosclerotic lesions displayed great
variations in cell type and distribution. The overlying endothelium was
intact to variable degrees, and -actin-positive SMCs appeared in
various stages of lesions, most frequently in advanced lesions (Figure 1)
. Cells expressing the macrophage
antigen identified by the RAM11 antibody were observed in all
atherosclerotic lesions. T lymphocytes identified by monoclonal
antibody L11/135 were also found in lesions (Figure 1)
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SAPK/JNK kinases are activated by dual phosphorylation of tyrosine
and threonine residues in response to stress stimuli or
mitogens.13-15
After activation, they translocate from
cytoplasm into the nucleus in cultured cells. We performed an
immunofluorescent analysis of atherosclerotic lesions using the
antibody recognizing the pan-SAPK/JNK and phosphorylated SAPK/JNK,
respectively. Intimal endothelium, media, and adventitia of normal
aortas showed very weak SAPK/JNK staining (Figure 2, a and d)
, while the lesion-covered
areas in intima from rabbits receiving a cholesterol-rich diet showed
increased immunostaining intensity (Figure 2, c, e, and f)
. Nonspecific
reactivity was minimal in the negative controls stained with a normal
mouse Ig (Figure 2b)
. The pattern of pan-SAPL/JNK and phosphorylated
SAPK/JNK staining in the lesioned aortas is shown in Figure 2, c, e, and f
. Heterogeneity of pan-SAPK/JNK and phosphorylated SAPK/JNK
staining became more evident in atherosclerotic plaques. Sites of
increased pan-SAPK/JNK and phosphorylated SAPK/JNK were mainly within
the cap and base regions of the atherosclerotic plaque (Figure 2, c, e, and f)
. Fatty streaks displayed elevated SAPK/JNK content in
subendothelial regions.
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, shows data representing double
immunostaining with antibodies against phosphorylated-SAPK/JNK (a and
c; green), -actin (b; red), and RAM11 macrophages (d; red).
Nonspecific staining was minimal in the corresponding negative controls
stained with a normal mouse Ig. Typical double-positive cells are
indicated by arrows, demonstrating a portion of SMCs and macrophages in
lesions overexpressing SAPKs/JNKs.
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Because of the increased intensity of immunostaining for
lesions, it would be interesting to determine SAPK/JNK1 protein levels
of atherosclerotic lesions. Protein extracts from tissues of normal
intima/media, lesioned intima, and media were analyzed by Western blot
analysis. Abundant SAPK/JNK1 proteins in atherosclerotic lesions were
observed (Figure 4A)
. SAPK/JNK proteins
in lesioned intima were significantly higher than intima/media of
control animals and media of cholesterol-fed rabbits after
normalization to the respect of actin contents (Figure 4C)
. To further
demonstrate higher SAPK/JNK1 protein levels associated with increased
SAPK/JNK1 activity, a kinase assay was performed using c-Jun as a
substrate. Figure
4B shows the results of an experiment examining
SAPK/JNK1 activities in the vessel wall, indicating elevated
SAPK/JNK1 activities in atherosclerotic lesions. When immunocomplexes,
obtained similarly as for the kinase assay, were subjected to Western
blot analysis, the results shown in Figure 4B
(lower panel)
demonstrated equal efficacy of the immunoprecipitation from protein
extracts of both normal vessels and atherosclerotic lesions and higher
levels of SAPK/JNK proteins in the lesions. Figure 4C
summarizes data
from two independent experiments, implicating that SAPK/JNK activities
were at low levels in control vessels and media of cholesterol-fed
rabbits, but increased three- to fivefold in atherosclerotic lesions.
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There is evidence that transcription factor p53 accumulates in
human atherosclerotic tissues.43,44
Immunofluorescence
staining confirmed high levels of p53 in the lesions induced by the
cholesterol-diet in rabbits (Figure 5)
.
Interestingly, the most p53+ cells showed an
elevation in pan-SAPK/JNK1 proteins, ie, co-localized in
atherosclerotic lesions (Figure 5, b and c)
. The distribution pattern
of p53 was similar to pan-SAPK/JNK1. In addition, both p53 (Figure 5d)
and SAPK/JNK1 (Figure 5e)
proteins in cells of arterial walls from
control rabbits were expressed at very low levels, if any. To further
verify p53 levels in lesions, protein extracts were analyzed by Western
blot using anti-p53 antibodies. Data shown in Figure 6
indicated that p53 protein content in
the lesions was higher than proteins from the normal intima and media
(Figure 6A)
. When SAPK/JNK1 was immunoprecipitated and analyzed with
Western blot, a 53-kd band was identified by anti-p53 antibody (Figure 6B)
, indicating that a complex of SAPK/JNK1 and p53 is present in
atherosclerotic lesions. These findings are concomitant with the
results obtained from in vitro studies using cultured
cells.22,23
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LDL is a main lipoprotein carrying and transporting
cholesterol in hypercholesterolemic rabbits induced by cholesterol
feeding. LDL can be oxidized in the arterial wall. To test whether LDL
and oxidized LDL can induce SAPK/JNK activation, vascular SMCs from
humans and rabbits were stimulated with human LDL and oxidized LDL and
the kinase assay was performed. As shown in Figure 8, A and B
, LDL and oxidized LDL (100
µg/ml) treatment resulted in significantly increased SAPK/JNK
activation in SMCs; kinetic analysis indicated that this response
occurred as early as 10 minutes after treatment (Figure 8, A and B)
,
with maximum levels (five- to eightfold greater than untreated control)
achieved 20 minutes after treatment, almost returning to basal levels
by 4 hours. To further establish the relationship between LDL or
oxidized LDL treatment and SAPK/JNK activity, we performed a
dose-response analysis of LDL- or oxidized LDL-induced SAPK/JNK
activation. As shown in Figure 8, C and D
, SAPK/JNK activities
increased in a dose-dependent manner between 5 and 400 µg/ml (LDL),
and reached higher levels at concentrations between 100 to 400 µg/ml.
To verify the efficacy of immunoprecipitation and the phosphorylation
of SAPK/JNK1 stimulated by LDL, the immunocomplexes, obtained similarly
as for kinase assays, were subjected to electrophoreses and blotted
with antibodies against phosphorylated SAPK/JNK (Figure 8E
, upper
panel) and pan-SAPK/JNK1 (Figure 8E
, lower panel). The results indicate
that oxidized LDL stimulated SAPK/JNK1 phosphorylation and did not
alter protein levels (Figure 8E)
. Taken together, these findings
demonstrate that LDL and oxidized LDL induced SAPK/JNK activation and
phosphorylation, which were strongly potentiated by oxidized LDL
compared to native LDL.
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indicates that LDL stimulated SAPK/JNK
activation in LDL receptor-deficient SMCs. No significant difference
between the two types of SMCs was found. It has been established that
heparin blocks LDL-receptor binding, but pretreatment of LDL with
heparin did not inhibit LDL-stimulated SAPK/JNK activation (Figure 9B)
.
When LDL binds to the receptors, the complexes are internalized and
degraded, which can be inhibited by pretreatment of cells with
chloroquine or NH4Cl. To further exclude the
involvement of LDL receptors, SMCs were pretreated with both reagents
and stimulated with LDL, again with no apparent influence (Figure 9C)
,
indicating LDL receptor-independent activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well known that LDL specifically binds to apoB/E receptors to deliver cholesterol to SMCs,49 but LDL-initiated signal transduction pathways leading to SMC gene expression are not fully elucidated. Recently, several groups reported that LDL and oxidized LDL activate extracellular signal-regulated kinases in cultured SMCs and macrophages.46,50-53 In the present study, we demonstrated, for the first time, that LDL and oxidized LDL markedly stimulate SAPK/JNK activation, and do not influence SAPK/JNK protein levels in cultured SMCs. LDL-induced SAPK/JNK activation was observed in both normal and Watanabe rabbit SMCs, which are LDL-receptor deficient, and this induction was not influenced by heparin, an inhibitor of LDL-receptor binding, indicating a process independent of classic LDL receptors.
As described above, oxidized LDL accumulation in the vascular wall is a
characteristic feature of atherosclerosis.54,55
Oxidized
LDL can be taken up into monocytes/macrophages to form foam cells via
scavenger receptors, eg, CD36.56
CD36 has been
demonstrated to be abundantly expressed in lipid-laden macrophages
around the core region of atherosclerotic lesions.57
Oxidized LDL-CD36 interaction leads to increased expression of CD11b,
CD18, cytokines such as IL-1 and TNF, and PDGF.58
Although
the mechanisms that transduce signals from oxidized LDL-CD36 binding to
the nucleus are not well defined, previous studies have suggested that
CD36 mediates nuclear factor 
B activation in response to oxidized
LDL.59
We found that LDL and oxidized LDL strongly
stimulate SAPK/JNK activation, independent of classic LDL receptors,
implicating the involvement of scavenger receptors. Therefore, CD36
activation may be involved in oxidized LDL-activated SAPK/JNK signal
pathways.
In addition to LDL, SAPK/JNK can be activated by cytokines, mechanical
force, oxidative stress, and growth factors present in high
concentrations in atherosclerotic lesions or involved in the
pathogenesis of atherosclerosis.1-12
These factors may be
partially responsible for elevated SAPK/JNK activities in lesions
observed here. After activation, SAPK/JNK kinases can phosphorylate or
activate several transcription factors, including c-Jun,
ATF2, Elk, and p53.16-23
ATF2 can dimerize not only with c-Jun, but also
with itself and some other members of the activation transcription
factor family, including ATF3, CREB, and nuclear
factor B. Elk-1, together with serum response factor, controls
transcription from the serum response element. These transcription
factors regulate gene expression, including matrix metalloproteinases,
adhesion molecule E-selectin, NO synthase, IL-8, and proliferating cell
nuclear antigen.1-3,60
These genes have been demonstrated
to play a key role in cell growth and cellular homeostasis in the
development of atherosclerosis. Thus, our findings of SAPK/JNK
activation in atherosclerotic lesions could be important to
understanding the mechanism controlling expression of these genes
during atherogenesis.
Apoptosis of SMCs and macrophages has recently been demonstrated in atherosclerotic lesions of humans as well as animal models.30-33,61-63 Oxidized LDL induces apoptosis of endothelial cells, SMCs, and macrophages depending on p53.27-29 Interestingly, isolated SMCs from human atherosclerotic plaques were shown to have a higher propensity for both spontaneous and induced apoptosis compared with SMCs from normal vessels.31 On the other hand, activation of MEKK1 (a SAPK/JNK upstream kinase) SAPK/JNK pathways is implicated in the initiation of apoptosis in other cell types in response to stress stimuli.64,65 In our study, we demonstrated coincidence of p53, pro-apoptotic protein, BAX, and BCL-XS with selective activation of SAPK/JNK in tissues of atherosclerotic lesions, indicating the involvement of SAPK/JNK in mediating apoptosis in vivo in response to hypercholesterolemia. It is conceivable that some cells in the lesions with higher SAPK/JNK activities and p53 proteins are subjected to apoptosis, during which, SAPK/JNK-mediated signal transduction pathways, ie, SAPK/JNK-activated p53, could be important in vivo.
As described above, LDL and/or oxidized LDL stimulate SMC proliferation as well as apoptosis, and both cell proliferation and apoptosis co-exist in atherosclerotic lesions. We believe that LDL and oxidized LDL can initiate several signaling pathways leading to (de)differentiation, proliferation, or apoptosis, and that the balance between the positive and negative signals in vascular SMCs is critical for maintaining homeostasis of the arterial wall. Further understanding of the mechanisms regulating SAPK/JNK activities could lead to new strategies for the prevention or treatment of atherosclerosis.
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Acknowledgements |
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Footnotes |
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Supported by grants P-12568-MED and P-13099-BIO (to Q. X.) from the Austrian Science Fund and 7919 (to Q. X.) from the Jubiläumsfonds of the Austrian National Bank. Dr. Hu is a recipient of an APART Award from the Austrian Academy of Sciences.
Accepted for publication March 3, 2000.
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K. Chen, J. A. Vita, B. C. Berk, and J. F. Keaney Jr. c-Jun N-terminal Kinase Activation by Hydrogen Peroxide in Endothelial Cells Involves Src-dependent Epidermal Growth Factor Receptor Transactivation J. Biol. Chem., May 4, 2001; 276(19): 16045 - 16050. [Abstract] [Full Text] [PDF]
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U. Mayr, M. Mayr, C. Li, F. Wernig, H. Dietrich, Y. Hu, and Q. Xu Loss of p53 Accelerates Neointimal Lesions of Vein Bypass Grafts in Mice Circ. Res., February 8, 2002; 90(2): 197 - 204. [Abstract] [Full Text] [PDF]
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