Strong Induction of Members of the Chitinase Family of Proteins in Atherosclerosis
Chitotriosidase and Human Cartilage gp-39 Expressed in Lesion Macrophages
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.
- Received July 23, 1998.
- Accepted August 28, 1998.
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.
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.
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.
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 α-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, 0.8× 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 5× 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, 2× 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, 2× SSC, and 15 minutes, 0.1× 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 β-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.
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.
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
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 α-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⇓).
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.
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⇓).
Chitotriosidase Activity in Vascular Tissue
Chitotriosidase activity can be assayed very sensitively using the fluorogenic substrate, 4-methylumbelliferyl-β-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⇑.
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.
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.
Barranger JA, Ginns, EI. Glucosylceramide lipidoses: Gaucher’s disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. New York: McGraw-Hill; 1989:1677–1698.
Hollak CEM, van Weely S, van Oers MHJ, Aerts, JMFG. Marked elevation of plasma chitotriosidase activity: a novel hallmark of Gaucher disease. J Clin Invest. 1994;93:1288–1292.
Renkema GH, Boot RG, Muijsers AO, Donker-Koopman WE, Aerts JMFG. Purification and characterization of human chitotriosidase, a novel member of the chitinase family of proteins. J Biol Chem. 1995;270:2198–2202.
Boot RG, Renkema GH, Strijland A, van Zonneveld AJ, Aerts JMFG. Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages. J Biol Chem. 1995;270:26252–26256.
Hakala BE, White C, Recklies, AD. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J Biol Chem. 1993;268:25803–25810.
Krause SW, Rehli M, Kreutz M, Schwarzfischer L, Paulauskis JD, Andreesen R. Differential screening identifies genetic markers of monocyte to macrophage maturation. J Leukoc Biol. 1996;60:540–545.
Renkema GH, Boot RG, Au FL, Donker-Koopman WE, Strijland A, Muijsers AO, Hrebicek M, Aerts JM. Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologous of family 18 glycosyl hydrolases secreted by activated macrophages. Eur J Biochem. 1998;251:504–509.
Ketcham CM, Roberts RM, Simmen RC, Nick HS. Molecular cloning of the type 5, iron-containing, tartrate resistant acid phosphatase from human placenta. J Biol Chem. 1989;264:557–563.
Giachelli CM, Bae N, Almeide M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. 1993;92:1686–1696.
Ikeda T, Shirasawa T, Esaki Y, Yoshiki S, Hirokawa K. Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta. J Clin Invest. 1993;92:2814–2820.
Shanahan CM, Cary NRB, Metcalfe JC, Weissberg PL. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest. 1994;93:2393–2402.
Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, 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. Arterioscler Thromb Vasc Biol. 1995;15:1512–1531.
Tso JY, Sun X-H, Kao T, Reece KS, Wu R. Isolation and characterization of rat and human GAPDH cDNAs. Nucl Acid Res. 1985;13:2485–2502.
Wilkinson D, Green J. In situ hybridization and the three-dimensional reconstruction of serial sections. In: Copp AJ, DL Cockroft, eds. Postimplantation Mammalian Embryos: A Practical Approach. Oxford, UK: Oxford University Press; 1990:155–171.
Shackelton LM, Mann DM, Millis AJT. Identification of a 38-kDa heparin-binding glycoprotein (gp38 k) in differentiating vascular smooth muscle cells as a member of a group of proteins associated with tissue remodeling. J Biol Chem. 1995;270:13076–13083.
Liaw L, Skinner MP, Raines EW, Ross R, Cheresh DA, Schwartz SM, Giachelli CM. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. J Clin Invest. 1995;95:713–724.
Ek-Rylander B, Flores M, Wendel M, Heinegård D, Andersson G. Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. J Biol Chem. 1994;269:14853–14856.
Semino CE, Specht CA, Raimondi A, Robbins PW. Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis. Proc Natl Acad Sci U S A. 1996;93:4548–4553.
Meyer MF, Kreil G. Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan. Proc Natl Acad Sci U S A. 1996;93:4543–4547.
Varki A. Does DG42 synthesize hyaluronan or chitin? A controversy about oligosaccharides in vertebrate development. Proc Natl Acad Sci U S A. 1996;93:4523–4525.
Savani RC, Wang C, Yang B, Zhang S, Kinsella MG, Wight TN, Stern R, Nance DM, Turley EA. Migration of bovine aortic smooth muscle cells after wounding injury: the role of hyaluronan and RHAMM. J Clin Invest. 1995;95:1158–1168.
Papakonstantinou E, Karakiulakis G, Roth M, Block LH. PDGF stimulates the secretion of hyaluronic acid by proliferating vascular smooth muscle cells. Proc Natl Acad Sci U S A. 1995;92:9881–9885.