© 1997 American Heart Association, Inc.
Articles |
Smooth Muscle Cells Express Granulocyte-Macrophage Colony-Stimulating Factor in the Undiseased and Atherosclerotic Human Coronary Artery
From the Department of Cell Biology and Ultrastructure Research, Institute for Arteriosclerosis Research, Münster, Germany (G.P., C.K., H.R.), and Imperial College School of Medicine, National Heart and Lung Institute, Royal Brompton Hospital, London, UK.
Correspondence to Dr Gabriele Plenz, Institute for Arteriosclerosis Research, Department of Cell Biology and Ultrastructure Research, Domagkstr 3, D-48149 Münster, Germany.
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Abstract |
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Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of a family of cytokines that regulate proliferation in macrophages and other types of cells, has been implicated in the inflammatory-fibroproliferative response of atherosclerosis. However, previous studies have been restricted to cultured cells and animal models. In the present study, we investigated GM-CSF expression in undiseased and atherosclerotic human coronary arteries at both the mRNA and protein levels. Dual in situ hybridization/cell-marking experiments demonstrated that subpopulations of intimal smooth muscle cells (SMCs) and endothelial cells express the cytokine in the histologically normal human coronary artery and that augmented expression occurs at these sites, and in macrophage accumulations and medial SMCs, in the atherosclerotic vessel. Corresponding data were obtained by in situ hybridization and reverse transcription–polymerase chain reaction and Northern analyses of cultured cells. Cultured human coronary arterial SMCs showed constitutive expression of GM-CSF in cells that had adopted an activated synthetic phenotype. Electron microscope immunocytochemistry revealed that GM-CSF is a protein localized in the cytoplasmic matrix of SMCs of both the undiseased and atherosclerotic vessel wall; extracellular matrix was largely unlabeled, with only occasional small patches of amorphous immunopositive material. The expression of GM-CSF by subpopulations of intimal SMCs in the undiseased artery and the marked upregulation of GM-CSF apparent in atherosclerotic lesions suggest roles for the cytokine in the cellular events underlying initiation and progression of the human atherosclerotic lesion.
Key Words: granulocyte-macrophage colony-stimulating factor • smooth muscle cells • human coronary artery atheroma
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Granulocyte-macrophage colony-stimulating factor, or GM-CSF, is a glycoprotein cytokine that was first characterized for its ability to stimulate progenitor hemopoietic cells to proliferate and differentiate into mature granulocytes and macrophages.1,2 GM-CSF was subsequently shown to have multiple effects on mature macrophages and lymphocytes, notably in immune activation and in stimulating proliferation,3–5 acting in concert with other members of the CSF family (M-CSF, granulocyte CSF, and IL-3) as a key mediator in inflammation and host defense.6–8 CSFs, including GM-CSF, are rapidly synthesized by a variety of cell types in response to injury, the ensuing accumulation of monocytes and macrophages and T lymphocytes in the tissue being the hallmarks of the inflammatory response.4,9–12
Atherosclerosis is essentially an inflammatory–fibroproliferative response of the arterial wall involving a complex set of interconnected events, including endothelial injury, phenotypic alteration of SMCs, accumulation of monocytes, macrophages, and T lymphocytes, and formation of lipid-laden foam cells.13–18 The cellular interactions underlying these events are regulated by cytokines and growth factors that are synthesized and released by the constituent cells of the arterial wall (endothelial cells, SMCs, macrophages, and T lymphocytes). Monocytes and macrophages are a major source of foam cells in the atherosclerotic plaque19; the accumulation and death of these cells lead to development of the lipid core, the classic feature of the lesion, which makes it prone to rupture and consequent life-threatening thrombosis.20,21 Key determining events in the pathogenesis of atherosclerosis are thus macrophage activation, proliferation, and survival, processes that are known to be regulated by CSFs.22–24
Studies on CSFs in atherosclerosis to date have
centered principally on M-CSF and the
macrophage,22,23 but indirect evidence
implicating GM-CSF as a critical player has steadily mounted. M-CSF and
GM-CSF are reported to lower plasma cholesterol levels in
humans and animals,25–27 and M-CSF enhances
uptake and degradation of acetylated LDL and increases
cholesterol esterification in human monocyte-derived
macrophages in vitro.28 A number of
cultured human cell types, including monocytes,
endothelial cells, and fibroblasts, have the capacity
to express GM-CSF in vitro,29–31 and GM-CSF
expression in human arterial SMCs in vitro is reportedly
inducible by inflammatory mediators such as IL-1 or
TNF-
.32 In atherosclerotic lesions of
cholesterol-fed rabbits, GM-CSF has been immunolocalized to
macrophages, with low levels reported in
endothelial cells and SMCs.24
Taken together, these findings point to potentially important roles for
GM-CSF in atherogenesis, but in the absence of any data on GM-CSF
expression in the human artery in situ, it has not previously been
possible to assess their significance in human coronary heart
disease.
We investigated GM-CSF expression, at both the mRNA and protein levels, in intact tissue from undiseased and atherosclerotic human coronary arteries. Our findings reveal that GM-CSF is expressed in defined subpopulations of cells in the undiseased artery and that marked upregulation occurs in atherosclerotic lesions. Importantly, subpopulations of SMCs are a potentially significant source of GM-CSF in the human coronary arterial wall.
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Methods |
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Patients, Samples, and Tissue Preparation
Human coronary artery specimens were obtained from the explanted hearts of male patients undergoing cardiac transplantation and from tissue discarded during surgical operations, as detailed in Table 1
. The protocol used for this study
fulfilled local ethical requirements. Undiseased arterial
samples were obtained from 7 patients (n=7), and atherosclerotic
samples were obtained from 18 patients (adaptive thickening, n=4;
preatheroma, type II and III lesions, n = 5;
atheroma and fibroatheroma, type IV and V
lesions, n=14; complicated lesion, n=4; and type VIII lesion,
n=3).33 Immediately on removal of the heart or
excision of surgical tissue, coronary artery samples from
proximal areas of the arteria coronaria dextra and arteria coronaria
sinistra were either placed in DMEM or prepared for structural studies.
The latter specimens were either fixed in 4%
paraformaldehyde fixative in PBS or placed in
cryoprotective medium (Cambridge Instruments) on small cork disks for
snap-freezing in liquid nitrogen and subsequent cryosectioning. The
frozen samples were stored at -80°C until required. The
paraformaldehyde-fixed samples were trimmed, and
selected areas containing plaques were taken for electron microscopy.
The remaining tissue was processed for paraffin embedding following
standard histological procedure. Two low-denaturation
preparation procedures were used for electron microscopy and
immunocytochemistry, cryoultramicrotomy according to the method of
Tokuyasu34 and embedding at low temperature in
the acrylic resin Lowicryl K4M following the protocol described by
Völker et al.35
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Isolation, Characterization, and Culture of Human
Arterial Smooth Muscle Cells
Endothelium was removed enzymatically from
aortic tissue or coronary arteries, and SMCs were subsequently
released by enzymatic digestion using 3 mg/mL
collagenase and 0.5 mg/mL
elastase.36–38 The cells were cultured in DMEM
with 20% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL
streptomycin, and 4 mmol/L L-glutamine. Cells
were routinely used between passages 3 and 6; for longer-term
cultivation experiments, cells up to passage 8 were used. All
components used in cell culture media were tested for endotoxin
contamination using the chromogenic Limulus
amebocyte lysate assay (endotoxin < 60 pg/mL, Endotect).
The purity of the SMC cultures was verified by immunohistochemistry
using specific SMC markers (see below). The decrease of the expression
of SMC-specific myosin during long-term cultivation experiments was
used as a marker for SMC "dedifferentiation," ie, change to the
synthetic phenotype.
Probes and Labeling Procedure
For hybridization, the recombinant cDNA clones hGM-CSF,
containing an insert complementary to the human GM-CSF mRNA (RD
Systems, Bad Nauheim, Germany), and G3PDH (Clontech, Heidelberg,
Germany), complementary to human
glyceraldehyde-3-phosphate dehydrogenase mRNA, were
used. For in vitro transcription, the GM-CSF cDNA was subcloned into a
pGEM vector (pGEM3Z, Promega Biotech, Madison, WI). The in vitro
transcription was performed according to the manufacturer's protocol
with modifications39 using digoxigenin-labeled
UTP.
Northern Blot Analysis
Total RNA was isolated from the cells according to the method of
Chirgwin et al.40 Northern blot analysis
was performed as previously described.41
Detection was performed with a modified detection protocol
(Boehringer Mannheim) and the chemoluminogenic substrate
CSP.
RT-PCR and Southern Blot Analysis
RNA from SMCs (undiseased coronary artery) was used. RNA
from human macrophages and human umbilical vein
endothelial cells was kindly provided by K. Peters
(Münster, Germany). One microgram of total RNA was reverse
transcribed after an initial denaturation step at 65°C for 5 minutes
in a total volume of 20 µL using 10 U/µL of Superscript II RNase
H- reverse transcriptase, 0.4 U/µL
ribonuclease inhibitor (MBI Fermentas GmbH), 25
ng/µL oligo[(dT)12–18], 0.5
mmol/L each of dNTP, 5 mmol/L DTT, 1x first-strand
buffer (50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L
KCl, and 3 mmol/L MgCl2) freshly
diluted from 5x stock at 37°C for 60 minutes. Samples were heated at
95°C for 5 minutes to terminate reverse transcriptase activity. Total
RT products were subsequently used for PCR amplification.
The RT products (20 µL) were brought to a volume of 50 µL containing 3 mmol of MgCl2, 0.2 mmol/L each of dNTP, 1x buffer (20 mmol/L Tris-HCl, pH 8.4, and 50 mmol/L KCl), 2.5 U of Taq polymerase, and 0.8 µmol/L of both the following upstream and downstream PCR primers for GM-CSF (DNA accession no. X03021, GenBank): CAAGCTTCTGTACAAGCAGGGCCTG (sequence location 1673 to 1690) and GCTCTAGATCCCAGCAGTCAAAGGGG (sequence location 2652 to 2669) (product size, 204 bp). Amplification was carried out in a Biometra UNO thermocycler after an initial denaturation at 94.5°C for 2 minutes for 35 cycles using the following temperature and time profile: denaturation at 94°C for 30 seconds, primer annealing at 58°C for 30 seconds, primer extension at 72°C for 20 seconds, and a final extension of 72°C for 15 minutes. Aliquots of the PCR reaction products (8 µL) were analyzed on a 1% ethidium bromide–stained agarose gel in 1 x Tris-borate electrophoresis buffer (90 mmol/L Tris-HCl, 90 mmol/L boric acid, and 2 mmol/L EDTA, pH 8.3) blotted on nylon filters and hybridized as described for Northern hybridization.
In Situ Hybridization
In situ hybridization was performed following methods modified
from those previously described.39 Enzymatically
released SMCs38 from coronary arteries
cultured on coverslips were fixed in 1%
paraformaldehyde; for tissues, paraffin-embedded
sections of material fixed in 4% paraformaldehyde and
cryosections were used. After rehydration, the slides were washed in
PBS and 5x Tris-EDTA buffer and treated with proteinase K (0 to 5.0
µg/mL 5x Tris-EDTA [50 mmol/L Tris-HCl, pH 8.0,
and 5 mmol/L EDTA]) for 10 minutes at room temperature.
The enzymatic digest was stopped with a Tris/glycine solution (50
mmol/L Tris-HCl and 0.2% glycine, pH 7.4). Cells and sections
were postfixed for 10 minutes in 1% or 4%
paraformaldehyde in PBS, washed in PBS, dehydrated in
an ascending series of ethanol, and air dried. Prehybridization was
performed in hybridization solution (50% formamide, 2x sodium
chloride/sodium phosphate/EDTA buffer [SSPE; 180 mmol/L
NaCl, 10 mmol/L
NaH2PO4, 1
mmol/L EDTA, pH 7.7], 10 mmol/L DTT, 2 mg/mL
herring sperm DNA, 200 µg/mL yeast tRNA, and 1 mg/mL
BSA) for 2 hours at 50°C. Before use, probes were heat denatured (10
minutes, 100°C) in hybridization solution. In situ hybridization was
performed either with 0.3 µg of digoxigenin-labeled cRNA-probe per
milliliter (antisense or sense strand of GM-CSF) or with hybridization
solution only at 50°C in a humidified chamber. For the lowest
stringency, buffers containing 2x SSC, and for the highest stringency,
0.1 x SSC, both at 50°C, were used.
Immunological Detection for In Situ Hybridization
Detection was performed with the aid of
anti-digoxigenin–alkaline phosphatase according to the manufacturer's
instructions (Boehringer Mannheim). The alkaline phosphatase
staining procedure was performed in the dark for 4 hours or overnight
using NBT (67.5 µg/mL) and X-PO4 (35
µg/mL) as substrates. After a final incubation in Tris-EDTA
buffer (10 mmol/L Tris-HCl, pH 8.0, and 1
mmol/L EDTA) for 5 minutes, coverslips or sections were mounted
with Kaiser's glycerine gelatin.
Immunohistochemical Identification of Cell Types Combined With In
Situ Hybridization
The alkaline phosphatase detection procedure for in situ
hybridization was followed by immunohistochemical detection of
macrophages (mouse anti-human CD 68, clone PG-M1; DAKO,
Hamburg, Germany), endothelial cells (rabbit anti-human
von Willebrand factor; Sigma Chemical Co, Heidelberg, Germany),
or SMC-specific myosin (SM-1 and SM-2 ABC
isoforms,37 mouse anti-human myosin, clone hSM-V,
Sigma) using the peroxidase Vectastain Elite ABC kit according to the
manufacturer's instructions. In addition, because of the possibility
that hSM-V antibody may cross-react with the nonmuscle myosin
variant,37 a second SMC marker, HHF35 (mouse
IgG1, Enzo Diagnostic Inc), which recognizes smooth muscle
/
actin, was used. As secondary antibodies, biotin-conjugated
goat anti-rabbit IgG or horse anti-mouse IgG (Vector Laboratories,
Burlingane, CA) was used. The peroxidase staining procedure was
performed for 20 to 45 minutes at room temperature using AEC (0.2
mg/mL 0.5 mol/L sodium acetate buffer, pH 5.2) as
substrate. The sections were counterstained according to standard
procedures with methylene green and mounted with Kaiser's glycerine
gelatin.
Postembedding Immunogold Electron Microscopy
Immunocytochemical labeling of the ultrathin frozen
sections and the sections of Lowicryl-embedded tissue was performed by
floating grids serially on 10- to 20-µL droplets placed on parafilm
sheets. Pretreatment to block nonspecific binding sites and to quench
aldehydes present on the section surface was carried out with 10%
FCS in PBS (pH 7.4) for 15 minutes (for cryosections) or with 1% BSA
in PBS (pH 7.4) for 15 minutes (for Lowicryl sections). The sections
were incubated with mouse monoclonal anti-human GM-CSF primary antibody
(dilution, 1:150 to 1:250; Genzyme Corp, Cambridge, MA) in 5% FCS/PBS
(cryosections) or 1% BSA/PBS (Lowicryl sections) for 1 hour, followed
by four 5-minute washes in PBS to remove the unbound primary
antibodies. Controls, in which the primary antibody was omitted, were
processed in parallel. Incubation with gold-labeled secondary
antibodies (12-nm colloidal gold AffiniPure goat anti-mouse IgG,
Dianova, Hamburg, Germany) was then carried out for 1 hour, followed by
four 5-minute washes in PBS and five 2-minute rinses in distilled
water. In some experiments, double immunogold labeling, using the
GM-CSF primary antibody and hSM-V (to mark SMCs) was done. In this
case, cryosections incubated with GM-CSF primary antibodies followed by
anti-mouse 12-nm gold-labeled secondary antibodies, as described above,
were then treated with goat anti-mouse antibodies (as a blocking step)
before serial incubation with hSM-V (dilution, 1:100; 1 hour) and 6-nm
gold-labeled anti-mouse secondary antibodies (dilution, 1:20; 30
minutes), with intervening washing steps similar to those used in
single labeling. Omission of hSM-V incubation served as a control.
Before examination, sections were stained with 2% methylcellulose and
0.2% uranyl acetate (cryosections) or in saturated aqueous uranyl
acetate and lead citrate (Lowicryl sections). The grids were examined
with a Philips 201 or 410 electron microscope operated at 60
kV.
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Results |
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Distribution of GM-CSF mRNA in the Undiseased Arterial Wall and in Atherosclerotic Lesions
We first compared the overall patterns of GM-CSF mRNA expression in undiseased and atherosclerotic arterial samples by in situ hybridization. Sections of undiseased vessel consistently revealed distinct GM-CSF mRNA-positive cells (Fig 1
, a and b). The hybridization signal was
most conspicuous in the superficial intimal region. Expression of
GM-CSF mRNA was apparent in the endothelium and in
cells that were irregularly scattered in a zone extending 25 to 50
µm beneath the endothelium. The
endothelial signal was not uniform, however; some
endothelial cells showed weak expression, and others
showed none at all. Occasional positive cells were also apparent in the
medial region adjacent to the intima, although here the overall levels
of signal were lower, and the major part of the media revealed no
expression (Fig 1, a and b). Negative controls using labeled sense RNA
were completely devoid of signal (Fig 1, c). The pattern of expression
that was illustrated, although showing minor variation between samples,
was consistently observed in sections from all patients
examined.
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The extent and pattern of GM-CSF mRNA expression in intimal thickenings
and early and advanced atherosclerotic lesions differed from those of
the undiseased vessel. Identical stages of lesion development in
different specimens displayed the same behavior in terms of GM-CSF mRNA
expression and distribution. GM-CSF mRNA-positive cells were observed
in the intima, media, and adventitia, although the distribution of
signal was not uniform and varied during lesion progression. The
endothelium of early lesions showed signal that
resembled that of undiseased vessels, with a small but distinct
increase apparent in advanced lesions (Figs 1
and 2). In arteries showing minimally
thickened intimas, GM-CSF mRNA signal was unaltered in the intima but
increased in the media, showing uniform distribution extending
throughout the entire medial layer (Fig 1, d). This GM-CSF mRNA signal
increased with further intimal thickening (Fig 1, e) and was even more
prominent in overt atherosclerotic lesions (type V) (Fig 2, a and b).
In overt lesions, GM-CSF mRNA-positive cells were observed scattered
throughout the entire intima. Conspicuous features were the presence of
small groups of particularly strongly positive cells concentrated
immediately beneath the endothelium and large foci of
positive cells deep in the intima, in the region of the plaque core
(Fig 2, a and b). In contrast to the undiseased artery, signal was
apparent over major regions of the media (Fig 2, b) and in the
adventitia, where conspicuous expression was apparent in the vasa
vasorum (Fig 2, c). Further changes in the expression pattern were
observed in the most advanced lesion categories (types VI and VIII). At
this stage, a decline in GM-CSF mRNA signal was observed in both the
medial and intimal SMCs (Fig 2, e and f). Distinct patches of high
signal intensity remained associated with the lipid-rich core (Fig 2, e) and thrombi (Fig 2, f).
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Immunohistochemical Identification of Cells Expressing GM-CSF
mRNA
Results from in situ hybridization, as described above, permit
localization of GM-CSF mRNA-positive signal within the tissue as a
whole, but the ability to correlate the signal with the specific cell
types of the lesion is limited. To enable unambiguous identification of
the cell types that express GM-CSF mRNA, we combined in situ
hybridization with immunohistochemistry using cell-type–specific
antibodies to permit simultaneous identification of
endothelial cells, SMCs, and macrophages (Figs 3 and 4).
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Fig 3 shows the results obtained with undiseased arteries. The
subendothelial GM-CSF mRNA expression was clearly
localized to a subpopulation of SMCs in the intima (Fig 3, a and b).
This localization was observable with both the HHF 35 and hSM-V SMC
markers. The signal in the media, adjacent to the intima, was also
confirmed to be of SMC origin. The GM-CSF mRNA expression attributed to
endothelial cells was confirmed with the
endothelial marker (Fig 3, c). Macrophages were
not observed in the intima of undiseased arteries but were observed in
the adventitia, where they were found to be GM-CSF mRNA negative (Fig 3, d).
Fig 4 presents results obtained on atherosclerotic lesions using
the combined in situ hybridization/cell-marking approach. Both SMCs and
macrophages were identified as GM-CSF mRNA-positive cells
within the intima. Examples of GM-CSF mRNA-positive SMCs beneath the
endothelium and scattered throughout the intima are
shown in Fig 4, a and c. GM-CSF mRNA-positive SMCs were localized to
these sites with both cell markers, indicating expression of both the
SMC-specific myosin and actin by these cells. The two SMC markers also
confirmed the expression of GM-CSF mRNA by medial SMCs (Fig 4, b). In
both the intima and the media, SMCs that did not express GM-CSF mRNA
were also common. A proportion of intimal GM-CSF mRNA-positive cells
that were not labeled by the SMC marker were demonstrated to be
macrophages (Fig 4, d through f). In the conspicuous
accumulations of GM-CSF mRNA-positive cells at the plaque base,
macrophages were the most abundant cell type (Fig 4, d and f),
although some expressing SMCs were also present, as demonstrated in
serial sections stained with each of the cell-type markers (Fig 4, e
and f). For both macrophages and SMCs, GM-CSF mRNA signal was
observed in both lipid-laden cells and cells that had not accumulated
lipid (not shown). Results with the endothelial marker
confirmed the presence of GM-CSF mRNA in endothelial
cells (not shown). Some GM-CSF mRNA-containing cells did not react with
any of the cell markers; such cells were observed in the vessel lumen,
directly attached to the endothelium, in the intima,
and in the adventitia and were presumed to represent invading
monocytes, granulocytes, T cells, and fibroblasts. High levels of
GM-CSF mRNA were observed in the endothelium and media
of the vasa vasorum.
A summary of the pattern of GM-CSF expression observed during the
different stages of lesion development is given in Table 2.
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GM-CSF Expression in the SMC: Cell Culture and Electron
Microscope Immunocytochemistry
To confirm the in situ hybridization/cell-marking results and to
investigate further the SMC as a GM-CSF-producing cell, we conducted
experiments to determine whether cultures of pure SMCs expressed GM-CSF
mRNA and whether GM-CSF protein was detectable at the electron
microscopic level in SMCs of arterial tissue.
In situ hybridization of cultured human SMCs, enzymatically released
from media explants after three passages, demonstrated that GM-CSF mRNA
was constitutively expressed in vitro, in both cells derived from the
undiseased artery and those from atherosclerotic arteries (Fig 5, a). RT-PCR analysis of the
constituent cell types of the normal vessel wall (Fig 5, b) revealed
that only SMCs and endothelial cells express GM-CSF
mRNA, both at comparable levels. GM-CSF mRNA was not detected in human
macrophages cultivated under normal cell culture conditions.
When cultured aortic SMCs were maintained for long periods (7 weeks,
seven passages), during which dedifferentiation to a more synthetic
phenotype occurs, a marked elevation in GM-CSF expression was
apparent (Fig 6).
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In immunogold-labeling studies at the electron microscopic level, SMCs
were readily identifiable by their ultrastructural features. Gold
labeling demonstrated that GM-CSF is a protein localized in the
cytoplasmic matrix of intimal SMCs of the human coronary
artery. In cells from undiseased arteries showing a contractile
phenotype (Fig 7, a), the
labeling density was uniform over a given cell but varied from one cell
to the next (12 to 40 gold particles/µm2).
Nuclei and mitochondria were devoid of label. The greater part of the
extracellular matrix contained no gold label (Fig 7, a and b), but
occasional patches of amorphous immunopositive material were detected.
Label in the extracellular matrix was never observed in association
with collagen fibrils (Fig 7, b and c). Double labeling with GM-CSF and
SMC (hSM-V) primary antibodies and gold markers of different diameters
confirmed the ultrastructural identification of SMCs (Fig 7, d).
Similar results were observed with ultrathin cryosections and Lowicryl
sections.
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SMCS of the synthetic phenotype (Fig 8, a and b) and foam cells of SMC origin
from diseased tissue (Fig 8, c) revealed a pattern of GM-CSF
localization similar to that of contractile-type cells, with the
protein localized uniformly throughout the cytoplasmic matrix (labeling
densities from 16 to 43 gold particles/µm2).
Label was entirely absent from the Golgi apparatus,
endoplasmic reticulum, vesicles, and other membrane-bound organelles
that are prominent features of the synthetic-state cell (Fig 8, a and
b); correspondingly, no label was present over lipid droplets,
lysosomes, and cholesterol crystals in SMC foam
cells (Fig 8, c). The extracellular matrix was largely devoid of label,
although, as in the undiseased tissue, isolated patches of labeling
were observed in association with amorphous noncollagenous material. In
no samples was there evidence of release of GM-CSF by exocytosis or of
endocytic uptake and degradation.
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The specificity of the labeling patterns was confirmed in sections of control undiseased and diseased tissue (not exposed to primary antibody) run in parallel, which were consistently devoid of gold label.
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Discussion |
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The principal novel findings of the present study are that GM-CSF is expressed in histologically normal and atherosclerotic human coronary arteries and that subpopulations of SMCs form an important source of this cytokine. These conclusions, which are based on comprehensive experimental data from in situ hybridization backed by PCR and Northern analyses, together with dual in situ hybridization/immuno–cell marking and electron microscopic immunocytochemistry, significantly extend findings of previous studies. Although GM-CSF expression has previously been reported in a range of cultured human cell types relevant to atherogenesis29–31 and in atheromatous aortic lesions of cholesterol-fed rabbits,24 cultured SMCs were reported to express GM-CSF only after stimulation with other cytokines,32 and in the rabbit model, no SMC GM-CSF production was detectable in control arteries.24 Our results, in contrast, indicate that GM-CSF is constitutively expressed in nonstimulated cultures of human coronary arterial SMCs and that intimal SMCs in the histologically normal as well as the diseased human coronary artery are significant producers of the cytokine. Although the prevalence of atherosclerosis makes it impossible to rule out the absence of incipient disease in the arterial samples classified as normal in our study, histological assessment provides a more rigorous test of absence from disease than do clinical, physiological, or symptomatic criteria, and the samples may therefore be regarded as typical of the disease-free state in the adult human population.
Comparison of our GM-CSF data from human atherosclerosis with that previously reported from the rabbit model24 must be interpreted with reference to arterial architecture. The human coronary artery, in contrast to the arteries of rabbits and other small mammals, characteristically has a thickened intima containing SMCs that differ in morphology and gene expression from SMCs of the media; these intimal cells characteristically show epithelioid or synthetic characteristics, particularly toward the luminal side of the vessel.42–44 It is in this specific subpopulation of cells, of which there is no counterpart in the normal rabbit artery, that particularly pronounced GM-CSF expression was detected in the present study. A GM-CSF–secreting subpopulation of SMCs in this location is strategically placed to exert a marked proatherogenic stimulus, augmenting that of the scattered GM-CSF–positive endothelial cells.
The presence of such a potential inbuilt proatherogenic stimulus in histologically normal coronary artery might seem at odds with the absence of overt disease in these normal control samples. However, our demonstration by electron microscopic immunocytochemistry of the undiseased arteries that GM-CSF is predominantly confined within the SMCs is consistent with a large potential reservoir of the cytokine, which may progressively be released as the lesion progresses. The localization of GM-CSF to the cytoplasmic matrix rather than to membrane-bound secretory organelles as in some cell types45 indicates that release of GM-CSF from the SMC is mediated not by classic exocytosis but by other mechanisms, such as structural damage to or blebbing of the plasma membrane or death and disintegration of the cell following apoptosis.46 The low quantity of GM-CSF labeled in the extracellular matrix in all samples is consistent with the known biological activity of the cytokine, whose effects are known to be mediated at extremely low doses, at the limits of detectability by immunocytochemistry.
In keeping with our in situ hybridization and immunocytochemical findings on histologically normal and atherosclerotic arterial tissue, in situ hybridization, RT-PCR, and Northern blot analyses of cultured SMCs (isolated from both sources) revealed comparable levels of expression of GM-CSF mRNA over three subcultivations, with markedly increased expression on dedifferentiation to an activated synthetic phenotype (ie, conditions corresponding to those prevailing in the atherosclerotic plaque). Apart from SMCs, our in situ hybridization studies of tissue sections demonstrate that the GM-CSF gene is expressed in some endothelial cells of the undiseased human coronary artery and that endothelial expression is increased in atherosclerotic lesions. Furthermore, foci of accumulating macrophages in the atherosclerotic lesion show especially abundant GM-CSF expression. Again, the observations on cultured cells accord closely with these data, with RT-PCR analysis of cultured endothelial cells revealing GM-CSF mRNA levels similar to those of SMCs.
The hypothesis that such multiple sources of GM-CSF could act as one of the key regulators of macrophage survival, proliferation, and accumulation in the atherosclerotic lesion has previously been suggested from animal model and cultured cell studies,24,32 and our present findings provide the first evidence of the applicability of this hypothesis to the human lesion. In contrast to M-CSF, which has a selective action on macrophage differentiation and is known to be expressed in rabbit and human atherosclerotic lesions,22–24 GM-CSF additionally affects the differentiation of T lymphocytes, neutrophils, and eosinophils47–50 and stimulates proliferation in a number of nonhemopoietic cell types.51 GM-CSF may therefore act as a polyfunctional regulator in atherogenesis, as part of a "CSF network" involving cytokine-stimulated mesenchymal cells and inflammatory leukocytes, analogous to that postulated for other inflammatory lesions.24,32,51
Within such a framework, GM-CSF production by the endothelium and subendothelial SMCs of the normal arterial vessel wall could play a role in initiation of the inflammatory-type response, with further stimulation of expression as oxidized LDL accumulates,31,52 the resultant activation of inflammatory cells subsequently providing a major source of the cytokine during the progression stage of the disease. This hypothesis is in agreement with our observation that normal cultivated macrophages do not express GM-CSF but that abundant expression occurs in the activated macrophages of the atherosclerotic vessel wall. Finally, intimal SMC-derived GM-CSF could also play an important role as an early stimulus to phenotypic transformation and migration of medial SMCs, and in highly fibrotic atherosclerotic lesions containing few macrophages, subsequent augmentation of these effects could occur by an autocrine mechanism alone.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (grants SFB 310 and SFB 223) and the British-German Academic Research Collaboration program (project 815). We thank K. Schlattmann, M. Opalka, and B. Milskemper for their technical expertise. Part of this work will be included in the doctoral thesis of C. Koenig.
Received April 25, 1997; accepted July 11, 1997.
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References |
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