© 1999 American Heart Association, Inc.
Vascular Biology |
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Modulates the Expression of Type VIII Collagen mRNA in Vascular Smooth Muscle Cells and Both Are Codistributed During Atherogenesis
From the Institute for Arteriosclerosis Research, Division of Cell Biology and Ultrastructure Research (G.P., S.R., C.K., J.R., H.R.), and the Departments of Cardiothoracic Surgery (M.C.D.) and Pathology (H.A.B.), Münster, Germany.
Correspondence to Gabriele Plenz, PhD, Institute for Arteriosclerosis Research, Division of Cell Biology and Ultrastructure Research, Domagkstr 3, D-48149 Münster, Germany. E-mail plenz{at}uni-muenster.de
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Abstract |
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Abstract—The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and type VIII collagen was studied in human arteries. GM-CSF and type VIII collagen were codistributed in all layers of the walls of nondiseased arteries and during early atherogenesis with up to type V lesions. The number of cells expressing both mRNAs increased during the development of advanced atherosclerotic lesions. Whereas type VIII collagen expression increased further in complicated lesions, GM-CSF was downregulated. During early atherogenesis smooth muscle cells (SMC) and endothelial cells were the principal GM-CSF and type VIII collagen mRNA-expressing cell types. In advanced lesions monocytes/macrophages also expressed the mRNAs. In complicated lesions the number of GM-CSF mRNA-expressing SMC was markedly reduced. In in vitro experiments transforming growth factor-ß1, platelet-derived growth factor, and GM-CSF, but not basic fibroblast growth factor, stimulated the expression of type VIII collagen mRNA by SMC. GM-CSF transiently stimulated type VIII collagen transcription. Thus GM-CSF is a prominent component of the regulatory network influencing collagen metabolism during atherogenesis. By modulating the synthesis of type VIII collagen in SMC, GM-CSF may influence the course of plaque development and may govern processes such as cell movement, plaque stability, and thrombus organization.
Key Words: extracellular matrix • remodeling • plaque development • endothelial cells • macrophages
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Introduction |
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Atherosclerosis, the underlying cause of coronary heart disease, is essentially a complex inflammatory-fibroproliferative response of the arterial wall. Atherogenic processes include vascular injury, infiltration of monocytes/macrophages, lipid accumulation, phenotypic alteration of smooth muscle cells (SMC), and reorganization of extracellular matrix (ECM).1 2 Although the arterial wall is composed to a large extent of SMC and their ECM products, endothelial cells (EC), infiltrating cells, and adventitial fibroblasts each represent only a small proportion of cells occurring in the vessel wall.3
Ninety percent of the total protein in the atherosclerotic plaque consists of collagens, ie, type I and type III collagen.4 5 Other members of the collagen family, type IV and type V collagen, probably play important roles in the processes of SMC phenotype modulation, vascular repair, and plaque stabilization.6 7 It is becoming increasingly clear that type VIII collagen is a key structural component of the vasculature. Type VIII collagen belongs to the group of short-chain collagens and forms 3-dimensional networks.8 The homology of type VIII collagen to type X collagen has been described.9
Type VIII collagen has been localized to the tunica media and tunica intima of elastic arteries.10 11 Its expression by the constituent cell types of the arterial wall, ie, SMC and EC, has been reported.12 13 In the rat balloon injury model it has been demonstrated to be an important component of the SMC response to injury and has been suggested to play a functional role in mediating migration of SMC.14 Type VIII collagen is markedly expressed in atherosclerotic arteries.15
Atherogenic processes are modulated by cytokines and growth factors, which are synthesized and released by the constituent cell types of the vessel wall.16 17 SMC have been shown to be capable of producing a number of cell mediators such as inflammatory cytokines, eg, colony-stimulating factors (CSF),16 and growth factors involved in ECM remodeling, eg, basic fibroblast growth factor (bFGF),4 platelet-derived growth factor (PDGF),18 and transforming growth factor-ß1 (TGF-ß1),4 which interact with mediators produced by the other vascular cell types.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been increasingly implicated as a critical player in atherogenesis.19 GM-CSF is a glycoprotein cytokine first characterized for its ability to stimulate progenitor hematopoietic cells to proliferate and differentiate into mature granulocytes and macrophages.20 21 Subsequently it has been shown to have multiple effects in immune activation and in stimulating proliferation,22 23 acting in concert with other members of the CSF family as a key mediator in inflammation and host defense.24 25 Although CSF, including GM-CSF, are rapidly synthesized by a variety of cell types in response to injury, the ensuing accumulation of monocytes/macrophages and T lymphocytes in the tissue constitutes the hallmark of the inflammatory response.23 26 27
It has been shown previously that type VIII collagen and GM-CSF are strongly expressed in atherosclerotic lesions.15 19 Apparent similarities in the distribution patterns of these molecules were observed. However, attempts to evaluate their potential codistribution and association have not been made.
This study is an in situ hybridization, immunohistochemical, and molecular biological assessment of the temporal and spatial distribution of GM-CSF and type VIII collagen in the nondiseased arterial wall and during the development of atherosclerotic lesions in human coronary arteries. Furthermore, we studied the effects of GM-CSF on the expression of type VIII collagen mRNA in cultured SMC. Our results demonstrate that GM-CSF and type VIII collagen are codistributed in the arterial wall and provide evidence that GM-CSF might play a part in regulating remodeling of the ECM during atherogenesis.
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Methods |
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Patients, Samples, and Tissue Preparation
Human coronary artery specimens and aortic tissue samples were obtained from explanted hearts of patients undergoing cardiac transplantation and from tissue obtained during cardiac surgery as detailed in Table 1
. The protocol was
approved by the local ethics committee. Specimens were classified
according to the recommendations of the American Heart
Association.28 29 More than one sample was obtained from
most patients. Samples obtained from 7 patients were classified
as nonatherosclerotic (n=7; without or very thin intimal layer; intima
consists of SMC and EC, neither monocytes and macrophages nor T
lymphocytes were detected immunohistochemically). Atherosclerotic
samples were obtained from 23 patients (adaptive thickening, n=5;
preatheroma [type II and III lesions]:, n=9;
atheroma and fibroatheroma [type IV and type V
lesions], n=14; complicated lesion [type VI lesion], n=6; type VII
lesion, n=2; and type VIII lesion, n=3). Tissue samples were prepared
for structural studies as previously described
elsewhere.19
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Cell Culture
Human SMC (n=4) were isolated and cultured as previously
described19 or obtained from BioWhittaker Inc. In brief,
endothelium was removed enzymatically from aortic
tissue, and SMC were subsequently released by enzymatic digestion. The
cells were cultured in Dulbecco's modified Eagle's medium with 20%
FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 4 mmol/L
L-glutamine. To evaluate the effect of GM-CSF on the
transcription of 
1(VIII) procollagen mRNA, cell culture experiments
were performed as follows: SMC were cultured to subconfluence and then
preincubated for 48 hours with Dulbecco's modified Eagle's medium
with 0.5% FBS. Preincubation was followed by incubation with
Dulbecco's modified Eagle's medium with 0.5% FBS containing GM-CSF
(1.0, 10, 100, and 500 pg/mL; Genzyme) for 35 minutes, 2 hours, 4
hours, and 24 hours. After incubation, cells were washed twice in PBS
and then lysed directly in the culture flasks by adding 4 mL
guanidinium isothiocyanate solution.
Peripheral blood monocytes were isolated from volunteer donors (n=3) by monocytapheresis, followed by elutriation and countercurrent centrifugation as previously described,30 and maintained in RPMI-1640 medium supplemented with 20% pooled human serum, 1 mmol/L sodium pyruvate, 20 mmol/L glutamine, and 1x essential amino acids (Gibco). Total RNA was isolated after 7 days and 14 days cultivation and 14 days cultivation followed by stimulation with lipopolysaccharide (LPS; 10 and 100 ng/mL) for 24 hours.
Human umbilical vein EC (n=3) were isolated from umbilical cords and cultured as described elsewhere.31 32 For RNA isolation, confluent monolayers were lysed directly in the culture flasks. Until isolation of total RNA, all RNA lysates were stored at -20°C.
The purity of SMC and EC cultures was verified by immunohistochemistry using SMC and EC type-specific antibodies. Monocytes were differentiated from macrophages by immunohistochemistry and type-specific monoclonal antibodies.
Labeling Procedure and Northern Blot Analysis
For hybridization, the recombinant cDNA clones,
phGM-CSF,19 containing an insert complementary to the
human GM-CSF mRNA (R&D Systems), pBSIIa1Col8, complementary to human
procollagen 1(VIII), and G3PDH (Clontech), complementary to human
glyceraldehyde-3-phosphate dehydrogenase mRNA, were
used. In vitro transcription was performed according to the
manufacturer's protocol with modifications using digoxigenin-labeled
UTP (Boehringer Mannheim).
Total RNA was isolated from human umbilical vein EC, SMC, and human monocytes/macrophages according to Chirgwin et al.33 Northern blot analysis was performed as previously described19 overnight at 72°C. Detection was performed using a modified protocol (Boehringer Mannheim) and the chemiluminogenic substrate 3-(4-methoxispiro{1,2-dioxetan-3,2'-(5' chloro)tricyclo[3.3.1.13,7]-decan}-4-yl) phenylphosphate.
To evaluate the relative expression, the luminographs on x-ray film
were scanned using a laser densitometer (Personal Densitometer,
Molecular Dynamics). Densitometric values of procollagen 1(VIII)
mRNA expression were corrected for G3PDH mRNA values. Corrected values
were normalized against the level of expression determined at 48 hours
of preincubation. Relative expression was expressed in mean values
(n=4) ±SD. Differences were assessed by Student's t test.
Probability values <0.05 were considered significant.
Reverse Transcription-Polymerase Chain Reaction
One microgram of total RNA from SMC, human
monocytes/macrophages, and human umbilical vein EC was reverse
transcribed (RT) using Superscript II RNase H-
according to the manufacturer's instructions (Gibco BRL; Life
Technologies GmbH). The PCR amplification of the GM-CSF product was
performed as previously described.19 The upstream and
downstream PCR primers for GM-CSF (DNA accession No. X03021, GenBank)
5'-GCAAGCTTCTGTACAAGCAGGGCCTG-3' (sequence location 1673 to 1690)
and 5'-GCTCTAGATCCCAGCAGTCAAAGGGG-3' (sequence location 2652 to
2669), product size 204 bp, were used.
For amplification of the 1(VIII) procollagen (DNA accession
No. X57527, GenBank) product, the upstream and downstream primers
5'-AGCTGTGATGATGCCCCCTACACCA-3' (sequence location 2212 to 2235) and
5'-TTACATGGGATACAATAAATATCC-3' (sequence location 1713 to 1734),
product size 522 bp, were used. RT products were amplified in a
solution containing 3 mmol/L MgCl2, 0.1
mmol/L each of dNTP, 1x buffer (20 mmol/L Tris-HCl, pH 8.4,
50 mmol/L KCl), 2.5 U of Taq polymerase (Gibco BRL),
and 1 µmol/L of each primer. Amplification was performed after
initial denaturation at 95°C for 5 minutes for 35 cycles using the
following temperature and time profile: denaturation at 95°C for 30
seconds, primer annealing at 62°C for 30 seconds, primer extension at
72°C for 30 seconds, and a final extension of 72°C for 15 minutes.
Aliquots of the PCR reaction products were analyzed using
standard agarose gel electrophoresis.
In Situ Hybridization
In situ hybridization was performed as
described19 either with 0.3 µg/mL digoxigenin-labeled
cRNA probe (antisense or sense strand of the GM-CSF and type VIII
collagen cDNA) or with hybridization solution only at 52°C. Detection
was done using the anti-digoxigenin alkaline phosphatase system
according to the manufacturer's instructions (Boehringer
Mannheim). The staining procedure was performed in the dark for 4 hours
or overnight using nitroblue tetrazolium salt (NBT; 67.5 mg/mL; Biomol)
and 5-bromo-4-chloro-3-indolyl phosphate (X-PO4;
35 mg/mL; Biomol) as substrates. Sections were mounted with Kaiser's
glycerin gelatin.
Immunohistochemical Identification of Cell Types
Cultured cell types were identified as follows: (1) for
EC, rabbit anti-human von Willebrand factor (Sigma); (2) for
SMC, mouse anti-human /
actin (HHF35; Loxo); (3) for
macrophages, mouse anti-human 25F9 (BMA); and (4) for
monocytes, mouse anti-human heterocomplex MRP8-MRP14 (27E10; BMA) were
used. As secondary antibody detection system we used donkey anti-mouse
and donkey anti-rabbit immunoglobulin conjugated to Cy3 (Chemicon).
The alkaline phosphatase detection procedure for in situ hybridization was followed by immunohistochemical detection of (1) macrophages (mouse anti-human CD 68, clone PG-M1; Dako); (2) EC (rabbit anti-human von Willebrand factor); or (3) SMC (HHF35), using the POD Vectastain Elite kit according to the manufacturer's instructions (Serva). Type VIII collagen was localized by using mouse anti-bovine type VIII collagen (C8; Medac). As secondary antibodies, biotin-conjugated goat anti-rabbit IgG or horse anti-mouse IgG (Vector Laboratories) were used. The peroxidase staining procedure was performed as described.19 Alternatively the immunofluorescence protocol was performed using as secondary antibody detection system donkey anti-mouse or donkey anti-rabbit immunoglobulin conjugated to Cy3 (Chemicon).
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Results |
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GM-CSF and Type VIII Collagen Are Expressed by Different Cell Types of the Arterial Wall
We studied GM-CSF and type VIII collagen mRNA-expressing cell types using combined in situ hybridization and immunohistochemistry and in vitro RNA analyses. In the arterial wall both GM-CSF and type VIII collagen mRNA were expressed by EC, SMC (Figure 1A
and 1B), and macrophages
(Figure 2A and 2B). However, not all
cells stained by the respective markers also expressed GM-CSF or type
VIII collagen mRNA.
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), and after 14 days of
culture and stimulation with endotoxin lipopolysaccharide
(LPS). Both GM-CSF and type VIII collagen mRNA were found in monocytes.
Macrophages expressed type VIII collagen mRNA only. Treatment
of macrophages with endotoxin (10 and 100 ng/mL) did not
markedly change the expression of type VIII collagen mRNA, whereas the
expression of GM-CSF mRNA was induced after treatment with endotoxin
(100 ng/mL). Arrowheads in B' indicate the 28S and 18S ribosomal RNA;
other abbreviations are as in Figure 1
The expression of GM-CSF and type VIII collagen mRNA was also
studied in cultured EC, SMC, and macrophages. As demonstrated
by RT-PCR, both mRNA were expressed basally in cultured EC (Figure 1A
') and SMC (Figure 1B'). Monocytes placed in culture
expressed GM-CSF mRNA (Figure 2A') after 7 days of cultivation,
but not after 14 days of cultivation when differentiated into
macrophages. Cultivated monocytes developed into
macrophages within 2 weeks of cultivation. Monocytes were
differentiated from macrophages by staining with the monocyte
marker 27E10 and the macrophage marker 25F9. However, in the
14-day-old monocyte-derived macrophages GM-CSF mRNA could be
induced by endotoxin activation. In comparison, type VIII collagen mRNA
was basally expressed both in monocytes and macrophages (Figure 2B'). Treatment with endotoxin did not markedly affect the
level of type VIII collagen mRNA in 14-day-old monocyte-derived
macrophages.
Co-Occurrence of GM-CSF and Type VIII Collagen
The occurrence of GM-CSF mRNA and type VIII collagen protein in
the same areas of atherosclerotic lesions was demonstrated using
combined in situ hybridization and immunohistochemistry. In nondiseased
arteries type VIII collagen was strongly expressed by EC, and faint and
homogeneous staining was observed throughout the entire
media (not shown). In early lesions (Figure 3), exemplary shown for a type III
lesion, type VIII collagen was predominantly located in the
endothelium and the luminal aspect of the intima
(Figure 3A). Some signal also occurred in other parts of the
intima and in the media. The distribution pattern of type VIII collagen
was directly reflected by the occurrence of GM-CSF mRNA-expressing
cells (Figure 3B). In advanced, type V lesions, both GM-CSF mRNA
and type VIII collagen are more widely distributed (Figure 4A and 4B). In areas adjacent to clusters
of GM-CSF mRNA-expressing cells, type VIII collagen was more strongly
deposited (Figure 4C and 4D).
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Expression of GM-CSF and Type VIII Collagen in Nondiseased Arteries
and During Early Atherogenesis
To follow the expression patterns of GM-CSF mRNA and type VIII
collagen mRNA during atherogenesis, comparative in situ hybridizations
were performed. Apparently nondiseased coronary artery segments
consistently contained distinct GM-CSF (Figure 5A) and collagen type VIII mRNA-positive
cells (Figure 5B). Expression of both mRNAs was apparent in the
endothelium and in cells that were irregularly
scattered beneath the endothelium. Positive cells were
occasionally observed throughout the entire media. However, the signals
were lower and the major part of the media was devoid of label.
Positively stained cells were also regularly found in the vessel lumen.
Compared with type VIII collagen mRNA, fewer cells expressed GM-CSF
mRNA. These patterns of expression were consistently observed
in sections from all the patients examined.
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In intimal thickenings (Figure 5C and 5D) and early
atherosclerotic lesions (Figure 5E and 5F), the extents and
patterns of GM-CSF (Figure 5C and 5E) and collagen type VIII
(Figure 5D and 5F) mRNA expression differed from those in the
nondiseased vessels. In these early stages both GM-CSF and type VIII
collagen mRNA were present in the endothelium, as
shown before for nondiseased arteries, in the intima, and in the media.
Positive cells were distributed throughout the entire medial layer. As
already mentioned for the nondiseased arteries, the overall expression
of GM-CSF mRNA was lower compared with that of type VIII collagen
mRNA.
Expression of GM-CSF and Type VIII Collagen in Advanced (Types IV
and V) and Complicated (Types VI through VIII) Atherosclerotic
Lesions
The number of GM-CSF and type VIII collagen mRNA-expressing cells
increased with further intimal thickening and was yet more prominent in
advanced (types IV and V) atherosclerotic lesions (Table 2). Figure 6 shows the typical distribution patterns
of type VIII collagen and GM-CSF mRNA in a type V lesion. Type VIII
collagen (Figure 6A) and GM-CSF (Figure 6B to 6D)
mRNA-positive cells were observed scattered throughout the entire
thickened intima. Furthermore, a characteristic feature of this type of
lesion was clustering of positive cells in large foci deep in the
intima, particularly in the plaque core (Figure 6B), and in
small groups immediately below the endothelium (Figure 6C). In addition, conspicuous signals were apparent over large
regions of the media (Figure 6D) and also in the adventitia,
especially in the vasa vasorum (Figure 7). It is noteworthy that the regions of
high GM-CSF expression mainly coincide with the regions of high type
VIII collagen expression. However, not all areas showing marked
accumulation of type VIII collagen mRNA-expressing cells coincide with
strong GM-CSF mRNA expression (Figure 6D).
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In complicated lesions (types VI, VII, and VIII), a remarkable
reduction of the number of expressing cells was observed both in the
media and intima. The media was almost devoid of label (not shown). As
shown in a type VIII lesion, label in the intima was confined to plaque
core structures (Figure 8A and 8C).
However, the expression of type VIII collagen mRNA remained high
(Figure 8B and 8D), both in the intima and in the media. GM-CSF
and type VIII collagen mRNA were likewise codistributed in clustered
cells in the intima. Despite the overall reduction of GM-CSF
mRNA-expressing cells, their distribution coincides with regions of
high expression of type VIII collagen mRNA.
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Mural thrombi are a special feature of the type VI lesions. GM-CSF mRNA
(Figure 9A and 9C) and type VIII collagen
mRNA (Figure 9B and 9D) were both found in organized mural
thrombi.
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The expression of GM-CSF and type VIII collagen mRNA by EC, SMC,
and macrophages was assessed by double-staining in situ
hybridization immunohistochemistry. Expressing cell types at different
stages of lesion development were identified by using cell
type-specific antibodies (Table 2).
In early lesions, GM-CSF and type VIII collagen mRNA both were expressed in SMC and EC. Macrophages expressing the mRNAs were found only sporadically. In advanced lesions, the number of SMC and macrophages shown to express GM-CSF and type VIII collagen mRNA increased. Nevertheless, EC, SMC, and macrophages that did not express GM-CSF or type VIII collagen mRNA were also common. Some of the GM-CSF and type VIII collagen mRNA-positive cells did not react with any of the cell markers. Such cells were observed in the vessel lumen, attached directly to the endothelium, in the intima, and in the adventitia. They were presumed to represent invading monocytes, granulocytes, T lymphocytes, and adventitial fibroblasts.
GM-CSF Stimulates the Expression of Type VIII Collagen in
Vascular SMC
To explore further the possibility whether type VIII collagen mRNA
expression can be modulated by GM-CSF, we assessed the expression of
type VIII collagen mRNA in GM-CSF–stimulated primary cultures of SMC.
We studied the effect of GM-CSF in comparison with several growth
factors known to influence the expression of ECM molecules, ie, PDGF,
TGF-ß1, and bFGF.
Expression of type VIII collagen mRNA increased
(P<0.05) after treatment with GM-CSF (1.65-fold,
165.13±10.64%), PDGF (1.3-fold; 130.7±8.22%) and TGF-ß1
(1.2-fold; 122.26±5.19%). Treatment with bFGF did not affect
(0.9-fold; 92.5±8.58%) the transcription of collagen type VIII mRNA
(Figure 10A). Time-course and
dose-response studies on the induction of type VIII collagen mRNA by
GM-CSF (Figure 10B) demonstrated a maximal response (1.7- to
1.9-fold) at 2 to 4 hours after treatment with 100 pg/mL of the
factor.
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Discussion |
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Type VIII collagen is a component of the normal arterial wall.10 Moreover, its expression is enhanced in response to injury14 and in atherosclerotic arteries.15 As we recently demonstrated increasing levels of GM-CSF during atherogenesis,19 we now studied the relation of GM-CSF to the processes of ECM remodeling occurring during the development of atherosclerotic lesions.
Key findings of the present study are that (1) cultured EC, SMC, and monocytes simultaneously express basal levels of GM-CSF and type VIII collagen mRNA, (2) although cultured macrophages generally express type VIII collagen mRNA, transcription of GM-CSF has to be induced, (3) GM-CSF and type VIII collagen codistribute in apparently nondiseased human arteries and atherosclerotic coronary arteries with lesions of different severity, and (4) GM-CSF transiently stimulates the expression of type VIII collagen mRNA by SMC. Our results provide evidence that GM-CSF, one of the key mediators of inflammation and host defense, is also involved in the process of ECM remodeling during atherogenesis.
Our histological studies demonstrate that in nondiseased arteries and intimal thickenings EC and SMC are the major cell types synthesizing GM-CSF and type VIII collagen. EC are directly exposed to atherogenic factors and blood cells.34 Stimulation of GM-CSF synthesis by EC after interaction with monocytes has been described recently.35 Reportedly, GM-CSF is also a potent chemoattractant for monocytes and macrophages.36 It has been suggested that migration of SMC is related to stimulated expression of type VIII collagen in response to cell mediators and injury.14 37 Thus, GM-CSF directly attracts cells or might influence their immigration into the intima through its effect on the vascular ECM.
Exactly as described for the response-to-injury model in rat, the early stages of lesion development in humans feature strong expression of type VIII collagen mRNA. At these early stages our data reinforce the observations of Bendeck et al14 and Sibinga et al37 in the rat balloon injury model. However, the rather profound expression of type VIII collagen in advanced lesions indicates an additional function. At this stage of lesion development matrix accumulates in the intima38 39 but SMC migration has not been suggested as a factor of importance. Thus, deposition of type VIII collagen potentially mediated by GM-CSF may alter the mechanical properties of the intima and contribute to the stability of the lesions.
Type VI to VIII lesions are characterized by an unchanged strong expression of type VIII collagen but a decreased number of GM-CSF–expressing cells,19 indicating additional factors regulating the expression of type VIII collagen. Noteworthy, codistribution is restricted to macrophage-rich areas. Macrophages are supposed to be responsible for matrix metalloproteinase-mediated destabilization of the plaque cap and thus for plaque rupture,40 as well as for matrix remodeling via cytokine-dependent mechanisms.41 Therefore one may speculate that GM-CSF–mediated changes in ECM expression may counterbalance macrophage-induced ECM destabilization. The occurrence of GM-CSF and type VIII collagen in organized thrombi is an additional strong hint that both play a part in tissue organization. A type VIII collagen network may substitute or stabilize matrix structures in a time-restricted manner and, in crosstalk with GM-CSF, promote cell infiltration during thrombus organization.
In vitro EC and SMC simultaneously express basal levels of type VIII collagen and GM-CSF mRNA. Cultured monocytes and macrophages both express type VIII collagen mRNA, although the level markedly decreases during differentiation of monocytes to macrophages. GM-CSF mRNA was markedly expressed in monocytes, but in macrophages only after activation. Thus the synthesis of type VIII collagen and GM-CSF mRNA in cultured monocytes/macrophages is related to their phenotype and, for GM-CSF, depends on the stage of activation. Our finding that GM-CSF transiently stimulates the expression of type VIII collagen mRNA by SMC supports the concept of GM-CSF as a potential regulator of type VIII collagen during the development of atherosclerotic lesions. This effect is even stronger than the stimulation after treatment with PDGF and TGF-ß1, both known to regulate the synthesis of type VIII collagen by SMC.14 37
Our studies indicate that GM-CSF and type VIII collagen are critical players during atherogenesis. In addition to the role of GM-CSF as mediator of inflammation and chemoattractant, GM-CSF might be involved in the processes of ECM remodeling. By regulating the synthesis of type VIII collagen in SMC, GM-CSF may influence processes of plaque development, which are related to plaque stability and tissue organization.
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Acknowledgments |
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The authors thank Brigitta Milskemper for her expert technical assistance and Marianne Opalka for her expert photographic work. This work was supported by the Deutsche Forschungsgemeinschaft. Part of this work will be included in the doctorate thesis of C. Koenig.
Received April 20, 1998; accepted December 17, 1998.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol. 1995;57:791–804.[Medline] [Order article via Infotrieve]
2. Wissler RW. Theories and new horizons in the pathogenesis of atherosclerosis and the mechanisms of clinical effects. Arch Pathol Lab Med. 1992;116:1281–1291.[Medline] [Order article via Infotrieve]
3. Campbell JH, Campbell GR. Cell biology of atherosclerosis. J Hypertens Suppl. 1994;12:S129–S132.[Medline] [Order article via Infotrieve]
4. Kennedy SH, Qin H, Lin L, Tan EM. Basic fibroblast growth factor regulates type I collagen and collagenase gene expression in human smooth muscle cells. Am J Pathol. 1995;146:764–771.[Abstract]
5. Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol. 1994;144:1348–1356.[Abstract]
6. Ramos KS, Weber TJ, Liau G. Altered protein secretion and extracellular matrix deposition is associated with the proliferative phenotype induced by allylamine in aortic smooth muscle cells. Biochem J. 1993;289:57–63.
7.
Liau G, Chan LM. Regulation of extracellular matrix
RNA levels in cultured smooth muscle cells: relationship to cellular
quiescence. J Biol Chem. 1989;264:10315–10320.
8.
Sawada H, Konomi H, Hirosawa K. Characterization of
the collagen in the hexagonal lattice of Descemet's membrane: its
relation to type VIII collagen. J Cell Biol. 1990;110:219–227.
9. Sutmuller M, Bruijn JA, de Heer E. Collagen types VIII and X, two non-fibrillar, short-chain collagens: structure homologies, functions and involvement in pathology. Histol Histopathol. 1997;12:557–566.[Medline] [Order article via Infotrieve]
10. Kittelberger R, Davis PF, Greenhill NS. Immunolocalization of type VIII collagen in vascular tissue. Biochem Biophys Res Commun. 1989;159:414–419.[Medline] [Order article via Infotrieve]
11. Jander R, Korsching E, Rauterberg J. Characteristics and in vivo occurrence of type VIII collagen. Eur J Biochem. 1990;189:601–607.[Medline] [Order article via Infotrieve]
12. Macbeath JRE, Kielty CM, Shuttleworth CA. Structure and organization of type VIII collagen. Biol Soc Trans. 1995;23:513S.
13.
Sage H, Trueb B, Bornstein P. Biosynthetic and
structural properties of endothelial cell type VIII
collagen. J Biol Chem. 1983;258:13391–13401.
14.
Bendeck MP, Regenass S, Tom WD, Giachelli CM, Schwartz
SM, Hart C, Reidy MA. Differential expression of alpha 1 type VIII
collagen in injured platelet-derived growth factor-BB–stimulated
rat carotid arteries. Circ Res. 1996;79:524–531.
15. Macbeath JR, Kielty CM, Shuttleworth CA. Type VIII collagen is a product of vascular smooth-muscle cells in development and disease. Biochem J. 1996;319:993–998.
16. Libby P, Sukhova G, Lee RT, Galis ZS. Cytokines regulate vascular functions related to stability of the atherosclerotic plaque. J Cardiovasc Pharmacol. 1995;25(suppl):S9–S12.
17.
Nilsson J. Cytokines and smooth muscle cells in
atherosclerosis. Cardiovasc Res. 1993;27:1184–1190.
18. Libby P, Warner SJC, Salomon RN, Birinyi LK. Production of platelet-derived growth factor-like mitogen by smooth-muscle cells from human atheroma. N Engl J Med. 1988;318:1493–1498.[Abstract]
19.
Plenz G, Koenig C, Severs NJ, Robenek H. Smooth muscle
cells express granulocyte-macrophage colony-stimulating factor
in the undiseased and atherosclerotic human coronary artery.
Arterioscler Thromb Vasc Biol. 1997;17:2489–2499.
20.
Burgess AW, Metcalf D. The nature and action of
granulocyte-macrophage colony stimulating factors.
Blood. 1980;56:947–958.
21.
Metcalf D. The granulocyte-macrophage
colony-stimulating factors. Science. 1985;229:16–22.
22. Metcalf D. The molecular control of cell division, differentiation commitment and maturation into haemopoietic cells. Nature. 1989;339:27–30.[Medline] [Order article via Infotrieve]
23. Metcalf D. Control of granulocytes and macrophages: molecular, cellular, and clinic aspects. Science. 1992;254:529–533.
24.
Clark SC, Kamen R. The human hematopoietic
colony-stimulating factors. Science. 1987;236:1229–1237.
25. Ölberg F, Botling J, Nilsson K. Macrophages and cytokine network. Transplant Proc. 1993;25:2044–2047.[Medline] [Order article via Infotrieve]
26. Bussolino F, Colotta F, Bocchietto E, Guglielmetti A, Mantovani A. Recent developments in the cell biology of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor: activities on endothelial cells. Int J Clin Res. 1993;23:8–12.
27. Hart MN, Fabry Z, Love-Homan L, Keiner J, Sadewasser KL, Moore SA. Brain microvascular smooth muscle and endothelial cells produce granulocyte macrophage colony-stimulating factor and support colony formation of granulocyte-macrophage-like cells. Am J Pathol. 1992;141:421–427.[Abstract]
28.
Stary HC, Bleakley Chandler A, Glagov S, Guyton JR,
Insull W Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler
RW. A definition of initial, fatty streak, and intermediate lesions of
atherosclerosis: a report from the Committee on
Vascular Lesions of the Council on
Arteriosclerosis, American Heart Association.
Arterioscler Thromb. 1994;14:840–856.
29.
Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S,
Insull W Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A
definition of advanced types of atherosclerotic lesions and a
histological classification of
atherosclerosis: a report from the Committee on
Vascular Lesions of the Council on
Arteriosclerosis, American Heart Association.
Circulation. 1995;92:1355–1374.
30.
Schmitz G, Fischer H, Beuck M, Hoecker KP, Robenek H.
Dysregulation of lipid metabolism in Tangier
monocyte-derived macrophages.
Arteriosclerosis. 1990;10:1010–1019.
31. Jaffe EA, Nachmann RL, Becker CG. Culture of human endothelial cells from umbilical veins. J Clin Invest. 1973;52:2745–2756.
32. Friedl P, Tatje D, Czapla R. An optimized culture medium for human vascular endothelial cells from umbilical cord veins. Cytotechnology. 1989;2:171–179.
33. Chirgwin JM, Przbyla AE, MacDonald E, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979;18:5294–5299.[Medline] [Order article via Infotrieve]
34. Chen CH, Henry PD. Atherosclerosis as a microvascular disease: impaired angiogenesis mediated by suppressed basic fibroblast growth factor expression. Proc Assoc Am Physicians. 1997;109:351–361.[Medline] [Order article via Infotrieve]
35.
Takahashi M, Kitagawa S, Masuyama J, Ikeda U, Kasahara
T, Takahashi Y, Furukawa Y, Kano S, Shimada K. Human monocyte
endothelial cell interaction induces synthesis of
granulocyte macrophage colony stimulating factor.
Circulation. 1996;93:1185–1193.
36. Wang JM, Colella S, Allavena P, Mantovani A. Chemotactic activity of human recombinant granulocyte-macrophage colony-stimulating factor. Immunology. 1987;60:439–444.[Medline] [Order article via Infotrieve]
37.
Sibinga NES, Foster LC, Hsieh CM, Perrella MA, Lee WS,
Endege WO, Sage EH, Lee ME, Haber E. Collagen VIII is expressed by
vascular smooth muscle cells in response to vascular injury. Circ
Res. 1997;80:532–541.
38. Wight TN. The vascular extracellular matrix. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. New York: Lippincott-Raven; 1996:421–440.
39. Ross R, Fuster V. The pathogenesis of atherosclerosis. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. New York: Lippincott-Raven; 1996:441–460.
40. Libby P, Geng YJ, Aikawa M, Schoenbeck U, Mach F, Clinton SK, Sukhova GK, Lee RT. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol. 1996;7:330–335.[Medline] [Order article via Infotrieve]
41. Rasmussen LM, Wolf YG, Ruoslahti E. Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor beta activity. Am J Pathol. 1995;147:1041–1048.[Abstract]
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