© 1999 American Heart Association, Inc.
Original Contributions |
Strong Induction of Members of the Chitinase Family of Proteins in Atherosclerosis
Chitotriosidase and Human Cartilage gp-39 Expressed in Lesion Macrophages
From the Departments of Biochemistry (R.G.B., T.A.E.A., B.E.A., G.H.R., J.M.F.G.A., C.J.M.V.) and Vascular Surgery (M.J.H.M.J.), University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
Correspondence to C.J.M. de Vries, PhD, Department of Biochemistry, University of Amsterdam, Academic Medical Center, K1-163, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail c.j.devries{at}amc.uva.nl
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Abstract |
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Abstract—Atherosclerosis is initiated by the infiltration of monocytes into the subendothelial space of the vessel wall and subsequent lipid accumulation of the activated macrophages. The molecular mechanisms involved in the anomalous behavior of macrophages in atherogenesis have only partially been disclosed. Chitotriosidase and human cartilage gp-39 (HC gp-39) are members of the chitinase family of proteins and are expressed in lipid-laden macrophages accumulated in various organs during Gaucher disease. In addition, as shown in this study, chitotriosidase and HC gp-39 can be induced with distinct kinetics in cultured macrophages. We investigated the expression of these chitinase-like genes in the human atherosclerotic vessel wall by in situ hybridizations on atherosclerotic specimens derived from femoral artery (4 specimens), aorta (4 specimens), iliac artery (3 specimens), carotid artery (4 specimens), and coronary artery (1 specimen), as well as 5 specimens derived from apparently normal vascular tissue. We show for the first time that chitotriosidase and HC gp-39 expression was strongly upregulated in distinct subsets of macrophages in the atherosclerotic plaque. The expression patterns of chitotriosidase and HC gp-39 were compared and shown to be different from the patterns observed for the extracellular matrix protein osteopontin and the macrophage marker tartrate-resistant acid phosphatase. Our data emphasize the remarkable phenotypic variation among macrophages present in the atherosclerotic lesion. Furthermore, chitotriosidase enzyme activity was shown to be elevated up to 55-fold in extracts of atherosclerotic tissue. Although a function for chitotriosidase and HC gp-39 has not been identified, we hypothesize a role in cell migration and tissue remodeling during atherogenesis.
Key Words: osteopontin • tartrate-resistant acid phosphatase • in situ hybridization • atherosclerotic lesion • human
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Introduction |
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Atherosclerosis is the pathological process in the vessel wall that eventually results in complete obstruction of blood flow (for review see Reference 11 ). The initiation of atherogenesis is considered to involve activation of endothelial cells, which facilitates monocyte infiltration into the vessel wall. These monocytes differentiate into macrophages, which accumulate lipids from the circulation and remain in the vessel wall, thereby becoming so-called foam cells. Subsequent migration and proliferation of smooth muscle cells (SMCs) from the media into the neointima is observed, probably induced by growth factors and cytokines secreted by the activated endothelial cells and macrophages.
A better understanding of atherogenesis requires a more precise characterization of the proteins secreted in the vessel wall by macrophages that are involved in this pathological process. For this reason we studied the occurrence of chitotriosidase in atherosclerotic vessels, an enzyme that has recently been shown to be expressed in lipid-laden macrophages in Gaucher disease (J.M.F.G. Aerts et al, unpublished data, 1997). This disorder is caused by an inherited deficiency in the lysosomal hydrolase glucocerebrosidase, resulting in accumulation of the lipid glucosylceramide in lysosomes of tissue macrophages.2 The multiorgan occurrence of lipid-laden macrophages in patients with Gaucher disease causes a variety of clinical symptoms such as hepatosplenomegaly and bone lesions. The abnormal macrophages of patients with Gaucher disease synthesize large amounts of chitotriosidase, resulting in several hundred-fold increased plasma enzyme levels.3 4 In some other inherited lysosomal storage disorders, especially sphingolipidoses such as Niemann Pick, GM1-gangliosidosis, and Krabbe disease, which involve accumulation of different lipids, more modest elevations in plasma chitotriosidase have been noted.5
Chitotriosidase was initially identified as an enzyme with catalytic activity toward the synthetic substrate chitotrioside.3 Later, chitotriosidase has been shown to exhibit chitinolytic activity toward the glucosaminoglycan chitin.6 On cloning of the full-length cDNA, the complete amino acid sequence became available, which revealed homology with the family 18 of glycosylhydrolases.7 A human homolog of chitotriosidase, human cartilage glycoprotein-39 (HC gp-39), was first identified in synovial fluid of patients with rheumatoid arthritis and has been shown to be synthesized by articular chondrocytes and synovial cells.8 This protein lacks enzymatic activity toward chitin and chitinlike substrates and has been proposed to be involved in tissue remodeling. It has been noted that HC gp-39 is also expressed by in vitro cultured, activated macrophages.9 10
We hypothesized that, analogous to Gaucher disease, lipid-laden macrophages present in the atherosclerotic vessel wall express chitotriosidase as well as HC gp-39. Therefore, we compared the expression of these genes with the expression of tartrate-resistant acid phosphatase (TRAP)11 12 13 and osteopontin,14 15 16 which are known to identify the entire population of activated macrophages and a subset of macrophages, respectively. Detailed analysis by in situ hybridization revealed that chitotriosidase, HC gp-39, and osteopontin are expressed in distinct subpopulations of infiltrated macrophages in atherosclerotic lesions. Clearly, phenotypic differences within macrophages in atherosclerotic lesions do exist. The potential physiological significance of the synthesis of members of the chitinase protein family in atherosclerosis is discussed in relation to the marked tissue remodeling that is associated with atherosclerosis.
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Methods |
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Human Tissue Samples
Human umbilical arteries were dissected from umbilical cords and stored at -80°C. Tissue samples from coronary arteries and aorta were obtained from heart transplantation recipients' hearts; apparently normal vascular material was dissected from cardiomyopathic hearts, whereas atherosclerotic coronary arteries were isolated from ischemic hearts. Apparently normal vascular tissue and early stages of atherosclerosis were obtained during organ transplantation, whereas atherosclerotic vascular tissue was obtained during vascular surgery. All tissues were fixed within 15 minutes of resection; tissue samples for in situ hybridization and immunohistochemistry were fixed in saline formalin and then paraffin-embedded, whereas for protein extraction the tissue was immediately frozen in liquid nitrogen. The characteristics of the specimens used in this study are summarized in Table 1
. All human specimens were
obtained with informed consent of patients or relatives according to a
protocol approved by the Medical Ethical Committee of the Academic
Medical Center.
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RNA Isolation and RNase Protection Assay
Total RNA was isolated with Trizol obtained from GIBCO BRL. For
the RNase protection assay [32P]UTP-labeled
antisense riboprobes were obtained by in vitro transcription of cDNA
fragments cloned in pGEM plasmids, containing T7 and SP6 RNA polymerase
transcription initiation sites (Promega). The following probes were
synthesized: HC gp-39: probe 248 nt, protected fragment 184 nt (bp 592
to 776)8 ; osteopontin: probe 264 nt, protected fragment
252 nt (bp 138 to 390)18 ; TRAP: probe 234 nt, protected
fragment 164 nt (bp 659 to 823)11 ; GAPDH: probe 114 nt,
protected fragment 84 nt (bp 480 to 564)19 ; and
chitotriosidase: probe 270 nt, protected fragment 199 nt (bp 970 to
1168).7 After linearization of the constructs riboprobes
were synthesized for 1 hour at 37°C in the RNA polymerase buffer,
supplied by the manufacturer (SP6 RNA polymerase [Promega], T7 RNA
polymerase [Stratagene]) and labeled with
[32P]UTP (Amersham). The probes were purified
by phenol-chloroform extraction, ethanol precipitation, and
electrophoresis on a 5% polyacrylamide gel containing 7 mol/L
urea and 45 mmol/L Tris-HCl-borate, pH 8.3, 1 mmol/L EDTA
(TBE) gel. For RNase protection, 4 µg of total RNA was hybridized for
18 hours with 105 cpm of
[32P]UTP-labeled riboprobe at 47°C in 20 µL
80% (vol/vol) formamide, 400 mmol/L NaCl, 40 mmol/L PIPES,
pH 6.4, and 1 mmol/L EDTA. After hybridization, nonhybridized RNA
was digested for 1 hour at 37°C in 350:1 Tris-HCl (pH 7.5), 300
mmol/L NaCl, 1 mmol/L EDTA, and 1 µg/mL RNase T1 (1300 U/µg,
GIBCO BRL). RNA was purified by proteinase K digestion,
phenol-chloroform extraction, and ethanol precipitation, and
subsequently electrophoresed on 5% polyacrylamide, 7 mol/L
urea, TBE gels. Quantification of the protected bands was performed
using a Molecular Dynamics PhosphorImager with Image Quant
software.
Immunohistochemistry
Immunohistochemical staining was performed on 5-µm paraffin
sections with the monoclonal antibody HAM56 (Dako) to detect
macrophages and the monoclonal antibody 1A4, directed against
SM 
-actin (Dako), to identify SMCs. The secondary goat anti-mouse
antibody was a biotin conjugate, which was subsequently detected with
streptavidin-peroxidase complexes (Dako). Peroxidase activity was
visualized with the substrate 3-amino-9-ethylcarbazole and
hydrogen peroxide.
In Situ Hybridization
Riboprobes were synthesized as described for the RNase
protection assay in the presence of [35S]-UTP
(Amersham). The length of the probes was as follows: chitotriosidase:
199 nt (bp 970 to 1168); HC gp-39: 374 nt (bp 402 to 776); osteopontin:
327 nt (bp 63 to 390); and TRAP: 494 nt (bp 329 to 823). Radiolabeled
probes were stored for up to 3 months in hybridization mix (40%
[vol/vol] formamide, 8% [wt/vol] dextran sulfate, 0.8x
Denhardt's, 0.5 mg/mL yeast tRNA, 4 mmol/L EDTA, 16 mmol/L
Tris-HCl [pH 8.0], 0.4 mol/L NaCl). Paraffin sections (5 µm)
of vascular tissue were mounted on 3-aminopropyltriethoxysilane-coated
slides. In situ hybridization was performed as described by Wilkinson
et al20 with minor modification. The sections were
pretreated with proteinase K (20 µg/mL) for 5 minutes, refixed in 4%
(vol/vol) paraformaldehyde, and treated for 10 minutes
with 0.25% (vol/vol) acetic anhydride in 0.1 mol/L triethanolamine (pH
8.0). Hybridizations were performed overnight at 50°C in 8 µL (0.5
µCi probe) per section under a coverslip in a moist chamber. After
hybridization, coverslips were removed in 5x SSC and 10 mmol/L
DTT at 50°C (30 to 60 minutes), followed by a high stringency wash
for 30 minutes at 65°C in 50% (vol/vol) formamide, 2x SSC, and
10 mmol/L DTT. RNase A digestion (20 µg/mL) was performed for 30
minutes at 37°C in 10 mmol/L Tris-HCl (pH 8.0), 5 mmol/L
EDTA, and 500 mmol/L NaCl. The high stringency wash was repeated,
followed by washings of 15 minutes, 2x SSC, and 15 minutes, 0.1x SSC.
After dehydration, autoradiography emulsion was applied
(Ilford G5 emulsion 1:1 diluted with 2% (vol/vol) glycerol). Slides
were developed in Kodak D19 after an exposure of 4 to 35 days, fixed in
Kodak UNIFIX, and counterstained with hematoxylin.
Monocyte Isolation and Macrophage Culture
Monocytes were isolated from citrated human blood by Percoll
density gradient centrifugation as described
previously.3 Cells were cultured in plastic Petri dishes
in RPMI 1640 (GIBCO BRL), supplemented with L-glutamine and
10% (vol/vol) FCS. At the indicated times, cells were harvested for
RNA isolation.
Protein Extraction and Chitotriosidase Enzyme Assay
Detergent-free protein extracts of the vascular tissues were
obtained by homogenization in 3 volumes of water
using an Ultra-turrax and centrifigation at 14 000g for 10
minutes at 4°C. The supernatant was stored at -20°C until use. The
protein concentration in the extract was determined with a
Bicinchoninic acid assay (Pierce) according to the instructions
of the manufacturer. Chitotriosidase activity was determined at 37°C
in a final volume of 125 µL with 22 µmol/L fluorogenic
substrate 4-methylumbelliferyl
ß-D-N,N',N''-triacetylchitotrioside
(Sigma) in McIlvain buffer (100 mmol/L citric acid, 200
mmol/L sodium phosphate, [pH 5.2]), containing 1 mg/mL BSA. The
reaction was stopped with 2 mL of 0.3 mol/L glycine, NaOH buffer (pH
10.6). Fluorescent 4-methylumbelliferone was measured with a
fluorimeter (Perkin-Elmer Corp) at 355 nm excitation and 460 nm
emission. The chitotriosidase activity of the extracts was expressed as
nanomoles substrate converted per hour per mg of protein. To
specifically inhibit the chitotriosidase activity a preincubation at
room temperature of the protein extract with polyclonal rabbit
antichitotriosidase3 was performed for 15 minutes
before addition of the substrate.
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Results |
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Expression of mRNA for Chitotriosidase, HC gp-39, Osteopontin, and TRAP in Cultured Macrophages
We have shown before that cultured macrophages express both TRAP, which is considered a marker for activated macrophages, as well as chitotriosidase, which is in accordance with the enhanced expression of this enzyme in abnormal macrophages in Gaucher disease.7 In this study we extended these analyses and determined the expression of the chitotriosidase homolog, HC gp-39, and of the extracellular matrix protein osteopontin in macrophages cultured for up to 22 days. Total RNA was isolated at different times (2 to 22 days), and the levels of mRNA expression were determined by RNase protection assay. The data of a typical experiment are shown in Figure 1A
through 1D. Two to 3 independent
batches of macrophages were cultured, and the data of the RNase
protection assays were quantified by PhosphorImager analysis
and summarized in Figure 1E through 1H. The genes assayed showed
different patterns of expression with time. Both TRAP (Figure 1A and 1E) and osteopontin (Figure 1B and 1F) were already
expressed at low levels in relatively undifferentiated
macrophages at day 2. The transient expression of HC gp-39
(Figure 1D and 1H) was first detected after 4 days, whereas
chitotriosidase expression (Figure 1C and 1G) was induced
relatively late after 7 to 9 days of macrophage culture. The
differences in kinetics of expression of these markers were observed in
macrophages obtained from different individuals and may
illustrate the subsequent phenotypic changes taking place during
maturation of in vitro cultured macrophages. These data
prompted us to determine the expression of chitotriosidase, HC gp-39,
osteopontin, and TRAP in the activated macrophages
present in atherosclerotic lesions.
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In Situ mRNA Expression of Chitotriosidase, HC gp-39,
Osteopontin, and TRAP in Atherosclerotic Lesions
To determine the potential colocalization of expression of
chitotriosidase, HC gp-39, osteopontin, and TRAP with
macrophages in vascular lesions, we performed radioactive in
situ hybridizations on sectioned material. The expression of these
genes was assayed in 21 specimens of vascular tissue derived from
different individuals ranging in age from 50 to 81 years (mean, 68
years). We analyzed atherosclerotic specimens derived from
different vascular origins and various stages of the disease: femoral
artery (4 specimens), aorta (4 specimens), iliac artery (3
specimens), carotid artery (4 specimens), and coronary artery
(1 specimen), as well as 5 specimens derived from apparently normal
vascular tissue (data summarized in Table 1
). No expression was
observed for each of these markers in the apparently normal specimens
(in situ hybridization data not shown). In Figure 2, results of the analysis of an
abdominal aortic aneurysm (Table 1, specimen 4) are
shown. Immunohistological analysis revealed the
presence of SMCs in the media (Figure 2A), which demarcates at
one side the adventitia (bottom of the picture), and on the other side,
the neointima, which extends up until the lumen of this
vessel. In the neointima, SMCs were observed (Figure 2A), as well as infiltrates of macrophages at the
luminal side of the lesion (Figure 2B). Furthermore, this
complex, advanced lesion contained a lipid core and a calcified area.
In a consecutive section the expression of chitotriosidase expression
was determined by in situ hybridization (Figure 2C), showing
expression of this chitinase in the macrophage infiltrate,
which is shown at a higher magnification in Figure 2D. Clearly,
not all macrophages expressed chitotriosidase, and the
expression of HC gp-39 was restricted to an even smaller subpopulation
of macrophages (Figure 2E). In this study we also
analyzed the gene expression of osteopontin, showing high
levels of expression of osteopontin in another subset of
macrophages (Figure 2F), which were in close vicinity of
the lipid core. TRAP was expressed throughout the complete
macrophage population, as expected (Figure 2G). In
contrast to the other genes assayed, osteopontin expression was not
restricted to the lesion macrophages, but was in addition
expressed in some of the neointimal SMCs (data not shown),
as has been published.14 15 16
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, macrophage; OPN, osteopontin. Original
magnifications x25 (A through C), x50 (D through G).
In Figure 3A and 3B, an overview is given
of an aortic specimen (Table 1, specimen 5) in which the media
was clearly visualized by high expression of SM -actin (Figure 3A), and a macrophage infiltrate was identified at the
luminal side of the lesion (Figure 3B). Magnification of part of
the macrophage infiltrate (Figure 3D) showed the absence
of any SMCs in this area (Figure 3C). All macrophages
identified by immunohistochemical staining exhibited low expression of
TRAP (Figure 3E), and a majority of the cells expressed
osteopontin at relatively high levels (Figure 3G). The
chitotriosidase gene was expressed in approximately half of the
macrophages (Figure 3F), whereas HC gp-39 mRNA was
localized only to the macrophages that had infiltrated deeper
in the lesion (Figure 3H).
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The expression pattern for each of these markers in an advanced
lesion of a carotid artery is shown in Figure 4 (specimen 9, Table 1). SMCs were
detected in the diminished media, in the deeper layers of the
neointima, and at the luminal side of the lesion, forming a
fibrous cap over the lipid core (Figure 4A). The
macrophages present in this lesion were localized near the
lipid core (Figure 4B). Figure 4C through 4H shows an
enlargement of the macrophage infiltrate in which no SMCs are
present (Figure 4C). TRAP was expressed in all
macrophages (Figure 4E), whereas osteopontin expression
was observed especially in those macrophages that line the
lipid core of the lesion (Figure 4G). Chitotriosidase mRNA
expression (Figure 4F) and HC gp-39 mRNA expression (Figure 4H) were observed only in different, smaller subsets of the
macrophages.
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In Figure 5, a detail of the
neointima of an early lesion in the iliac artery of a
50-year-old organ donor (specimen 15, Table 1), in which both
SMCs and macrophages were observed, is shown (Figure 5A and 5B). TRAP expression (Figure 5C) again colocalized exactly
with the macrophages identified by immunohistochemical staining
(Figure 5B), which was also the case for osteopontin (Figure 5E). Chitotriosidase expression was restricted to only a
minority of the macrophages and was relatively low (Figure 5D), whereas in situ hybridization with the HC gp-39–specific
probe showed high expression of this gene in a relatively high number
of macrophages (Figure 5F).
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Chitotriosidase Activity in Vascular Tissue
Chitotriosidase activity can be assayed very sensitively
using the fluorogenic substrate,
4-methylumbelliferyl-ß-D-N,N',N''-triacetylchitotrioside.
To correlate the presence of chitotriosidase-encoding mRNA in
atherosclerotic tissue with chitotriosidase activity, we prepared
extracts from apparently normal and atherosclerotic tissue and assayed
for chitotriosidase activity. The amount of activity was normalized to
the total protein concentration in the extracts (Table 2). To determine the specificity of the
assay, we simultaneously preincubated the extracts with a
polyclonal antibody directed against chitotriosidase that is known to
fully inhibit its enzymatic activity. In this study we assayed extracts
of human umbilical cord artery and extracts of aorta and
coronary arteries from nonischemic,
cardiomyopathic, transplantation recipient hearts to
determine the chitotriosidase expression in apparently normal tissue.
These vessels were judged as apparently normal on the basis of
macroscopic examination and did not show significant macrophage
content on immunohistochemical analysis with the
macrophage-specific antibody. The normal vessel wall
extracts contained very low chitotriosidase activity (0.5 to 2.1
nmol/mg per hour; data summarized in Table 2). Atherosclerotic
tissue, obtained from different arteries of different individuals
during vascular surgery, was analyzed and contained
chitotriosidase activities of 4.9 to 27.4 nmol/mg per hour. In
conclusion, the chitotriosidase activity was increased up to 55-fold in
atherosclerotic tissue compared with normal vessels. The wide range of
activities measured in the lesions reflects the diversity of the
resected material with respect to the number of macrophages
actually expressing chitotriosidase, as is illustrated in Figures 2 and 3 versus Figures 4 and 5.
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Discussion |
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A key event in the initiation of atherosclerosis is the augmented infiltration of monocytes into the vessel wall and their subsequent differentiation from macrophages into lipid-laden foam cells. In this study we revealed the expression of 2 new markers of activated macrophages in the human atherosclerotic plaque: the chitinolytic enzyme chitotriosidase6 7 and HC gp-39, both members of the chitinase family of proteins.8 We show for the first time a strong induction of chitotriosidase and HC gp-39 expression in subpopulations of macrophages associated with human atherogenesis.
Distinct kinetics of mRNA expression were observed for chitotriosidase and HC gp-39 in in vitro cultured macrophages. In addition, we have shown dissimilar differential expression of osteopontin and the macrophage marker TRAP in these cultures. From these data we could not predict the expression pattern of each of these genes in atherosclerosis, as the lifespan of a macrophage in the atherosclerotic plaque is expected to be much longer than 4 weeks, the latest time tested in this in vitro study. Furthermore, (un)identified factors expressed during atherogenesis but lacking in the in vitro cultures may influence the maturation and differentiation of macrophages trapped in lesions.
In our in situ hybridization experiments we have shown expression of the marker for lysosomal activity, TRAP, in each macrophage present in 21 different vascular specimens derived from distinct arteries and multiple stages of the disease. Expression of osteopontin was observed both in subsets of macrophages that line lipid cores, and in some neointimal SMCs, as has been published.14 15 16 The gene expression of chitotriosidase and HC gp-39, however, is restricted to relatively small, unique groups of macrophages, exemplifying the phenotypic variation among macrophages in the atherosclerotic lesion. More specifically, in early lesions, obtained from organ donors, only a minority of the macrophages expressed chitotriosidase, whereas HC gp-39 and osteopontin were expressed more abundantly.
Recently, the porcine homolog of HC gp-39, gp38 k, has been presented as a marker for highly differentiated, in vitro cultured porcine SMCs.21 In a related study we observed expression of HC gp-39 in cultured human SMCs derived from neonatal umbilical cord arteries, cells that are known to maintain the characteristics of differentiated SMCs in vitro (C.J.M. de Vries et al, unpublished data, 1997). Unfortunately, no data are available on the expression pattern of HC gp-39 in the porcine vessel wall. The in situ hybridizations performed in this study, however, revealed the manifest expression of HC gp-39 in subpopulations of human macrophages, without clear expression in the medial SMCs. HC gp-39 expression in normal, medial SMC was not detected with in situ hybridization, which could be because of the limited sensitivity of the in situ hybridization assay.
For osteopontin, both its expression in the human atherosclerotic plaque and its potential role in SMC adhesion and migration have been well documented.22 Here we present colocalization in vascular lesions of cells expressing osteopontin and cells expressing TRAP. TRAP has been reported to specifically dephosphorylate osteopontin, decreasing the functional activity of osteopontin in the extracellular matrix, where it is involved in osteoclast adhesion.23 It is tempting to propose that TRAP can modulate the adhesion of SMCs in the atherosclerotic plaque by modifying the extent of phosphorylation of osteopontin, thereby facilitating subsequent migration.
The expression of chitotriosidase mRNA in atherosclerotic tissue
correlated with the presence of chitotriosidase activity in extracts of
vascular tissue (Table 2). We were interested in determining
whether chitotriosidase protein and consequently enzyme activity were
also enhanced in serum of individuals suffering from
atherosclerosis. Preliminary experiments, in which the
chitotriosidase activity was determined in serum of patients with
Familial Hypercholesterolemia who had clinical
symptoms of atherosclerosis, did not show a prominent
elevation compared with normal individuals (data not shown). It should
be emphasized that the average chitotriosidase activity in serum shows
a mild, but significant, age-dependent increase in the general
population.5 It cannot be excluded that the age-dependent
elevation in chitotriosidase is at least partially caused by ongoing
accumulation of lipid-laden macrophages during the gradual
progression of atherosclerosis with aging.
The exact mechanism underlying the induction of chitotriosidase and HC gp-39 expression in macrophages is unknown. The composition of the lipids accumulating in in vitro cultured macrophages and in macrophages trapped in the atherosclerotic lesion is currently being investigated. The goal of these studies is to identify the specific signal that is crucial for the initiation of expression of chitinase-like genes in macrophages.
We can only speculate on the physiological role of chitotriosidase and HC gp-39 in atherogenesis, because no human endogenous chitinlike substances are known at present. Recently, a vertebrate chitin synthase has been identified, which is supposed to create short chitin stretches that are essential to initiate hyaluronan synthesis.24 25 26 The glucosaminoglycan hyaluronan is expressed in the extracellular matrix of the injured vessel wall and has been reported to be involved in SMC proliferation and migration.27 28 29 Possibly, chitotriosidase and HC gp-39 recognize hyaluronan (precursor) as a substrate and interfere with its synthesis, which could affect local hyaluronan concentrations and consequently influence the extent of cell migration in the injured vessel wall.
In summary, macrophages in atherosclerotic plaques form a heterogeneous group of infiltrated cells that all express TRAP. In this study phenotypic differences were visualized by specific expression patterns of osteopontin, chitotriosidase, and HC gp-39 in distinct subpopulations of lesion macrophages. We propose that involvement of each of these proteins in modulation of the extracellular matrix in the vessel wall may affect cell adhesion and migration during the tissue remodeling processes that take place during atherogenesis.
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Acknowledgments |
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This work was supported by the Molecular Cardiology Program of the Netherlands Heart Foundation (grant M93.007). We acknowledge the cooperation of the Department of Vascular Surgery of the Academic Medical Center in collecting the human vascular specimens and the technical assistance of A. Strijland. We also thank Drs. H. Pannekoek, J. Jansen, and A.J.G. Horrevoets for critically reading the manuscript.
Received July 23, 1998; accepted August 28, 1998.
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