(American Journal of Pathology. 2001;158:1465-1472.)
© 2001 American Society for Investigative Pathology
Cartilage-Specific Matrix Protein Chondromodulin-I Is Associated with Chondroid Formation in Salivary Pleomorphic Adenomas
Immunohistochemical Analysis
Kimihide Kusafuka*,
Yuji Hiraki
,
Chisa Shukunami,
Akira Yamaguchi
,
Teruo Kayano§ and
Tamiko Takemura*
From the Department of Pathology,*
Japanese Red Cross
Medical Center, Tokyo; the Department of Molecular Interaction and
Tissue Engineering,
Institute for Frontier
Medical Sciences, Kyoto University, Kyoto; the Department of Oral
Pathology,
School of Dentistry, Nagasaki
University, Nagasaki; and the Section of Forensic
Dentistry,§
Division of Public Health,
Department of International Health Development, Graduate School, Tokyo
Medical and Dental University, Tokyo, Japan
 |
Abstract
|
|---|
Chondromodulin-I (ChM-I) is a novel cartilage-specific matrix
protein. In the growth plates of the long bones, ChM-I was
shown to be expressed in mature to upper hypertrophic
chondrocytes, and to be deposited in the cartilage matrix. As
ChM-I strongly inhibits angiogenesis, cartilage is avascular.
Also, ChM-I has bifunctional activity against chondrocyte
proliferation. On the other hand, pleomorphic adenomas of the
salivary glands frequently have chondroid elements. To elucidate the
relationship between chondroid formation and hypovascularity in
salivary pleomorphic adenomas, we immunohistochemically
examined the expression and localization of ChM-I in 35 cases of this
tumor. ChM-I was immunolocalized to the lacunae in the chondroid
elements of pleomorphic adenomas (100%). Type II collagen and aggrecan
were immunolocalized throughout the matrix around lacuna cells of the
chondroid element (100%, 91.7%), and ChM-I was
infrequently immunolocalized to the spindle-shaped myoepithelial cells
in the myxoid element (37.5%). Fibroblast growth factor-2 was strongly
immunolocalized to the lacuna cells in the chondroid element
(100%), among the neoplastic myoepithelial cells in the myxoid
elements (96.9%), and on the basement membranes around the
solid nests of neoplastic myoepithelial cells (71.4%). Although CD34
is a marker of endothelial cells, CD34 was expressed in the
endothelial cells in only a few areas around the epithelial elements
and in the fibrous element of pleomorphic adenomas. No signals for CD34
were observed in chondroid elements in pleomorphic adenomas
(P < 0.001), but a few signals were seen
in the myxoid elements (P < 0.05). These findings
suggested that lacuna cells and neoplastic myoepithelial cells
expressed ChM-I, and that this molecule may play an important
role in hypovascularity and chondroid differentiation in pleomorphic
adenoma. In conclusion, pleomorphic adenoma expressed
ChM-I, which is involved in hypovascularity and chondroid
formation in this type of tumor.
|
Introduction
|
|---|
Pleomorphic adenoma of the salivary
glands is characterized by the so-called "mixed" appearance of
epithelial and mesenchymal-like elements. In previous studies,
mesenchymal-like elements including chondroid and myxoid elements were
shown to be related to neoplastic myoepithelial cells migrating into
the stroma. Recently, we demonstrated that bone morphogenetic proteins
(BMPs) were associated with chondroid formation in pleomorphic
adenoma.1,2
Also, we reported that co-expression of
fibroblast growth factor (FGF)-2 and FGF receptor-1 in the lacuna cells
in chondroid elements inhibited ossification of the chondroid
elements.3
Although FGF-2 is a strong angiogenic factor,
pleomorphic adenomas are hypovascular tumors and there were not any
capillaries in the chondroid elements of this type of tumor.
Chondromodulin-I (ChM-I), a cartilage-specific noncollagenous matrix
protein, was extracted and cloned from bovine cartilage.4
Recently, ChM-I has been reported to be a strong inhibitor of
angiogenesis, responsible for the avasucular nature of
cartilage.5,6
In the growth plates of the long bones,
ChM-I mRNA was expressed in the proliferating to the upper hypertrophic
chondrocytes and its product was deposited in the interterritotrial
matrix around the lacunae.6
The human ChM-I gene was
recently cloned.7
The findings presented here indicated that ChM-I, a strong
angio-inhibitor, may be expressed in chondroid elements of salivary
pleomorphic adenomas. We examined expression and localization of ChM-I,
in comparison with localization of FGF-2 and/or density of
CD34-positive endothelial cells, in salivary pleomorphic adenomas using
immunohistochemical methods.
|
Materials and Methods
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Antibodies
Anti-ChM-I polyclonal antibody was raised in a rabbit against
mature recombinant human ChM-I protein.7
On Western
blotting analysis, this antibody revealed a single diffuse band of 25
kd. Anti-CD34 monoclonal antibody (cat. no. 1185; Immunotech,
Marseille, France), anti-type II collagen monoclonal antibody (clone
II-4C11; Fuji Yakuhin Kogyo, Toyama, Japan), and anti-FGF-2 polyclonal
antibody (22-97-0175; RD Laboratorien, Herrsching bei Munchen, Germany)
were purchased from the sources shown. These antibodies were diluted
1:1, 1:500, and 1:1,000, respectively. Specificities of anti-type II
antibody and anti-FGF-2 (basic FGF) antiserum were confirmed
previously.1-3
Anti-aggrecan polyclonal antibody, a kind
gift from Dr. T. Yada (Institute for Molecular Science of
Medicine, Aichi Medical University, Nagoya,Japan), was raised
against rat cartilage aggrecan purified from 1-week-old rat tibial
cartilage as previously described.8
This antiserum
against rat aggrecan recognized mouse and human aggrecan core protein
on enzyme-linked immunosorbent assay and Western blotting analysis and
cross-reacted with human aggrecan.
Tissues
Thirty-five pleomorphic adenoma cases were chosen from the
pathology files of the Japanese Red Cross Medical Center, Tokyo, Japan,
from the period 1986 to 1998. The tubulo-glandular structures and
mesenchymal-like stromas of pleomorphic adenomas are summarized in
Table 1
. Twenty specimens included normal
salivary gland tissues. Two neonatal vertebral tissue, one enchondroma
tissue, two placenta tissue, and two tracheal cartilage specimens were
used as controls. These specimens were fixed in 10% buffered formalin,
routinely processed, and embedded in paraffin wax. Sections 3-µm
thick were then prepared.
Immunohistochemistry
Paraffin-embedded sections were deparaffinized in xylene and
rehydrated. For ChM-I staining, deparaffinized sections were first
pretreated with 0.1% hyaluronidase (H6254; Sigma Chemical Co., St.
Louis MO) in acetate buffer, pH 5.0, for 45 minutes at 37°C, followed
by two washes with phosphate-buffered saline (PBS), and then they were
treated with 0.4% pepsin (P-6887; Sigma Chemical Co.) in 1.0 N HCl
solution for 45 minutes at 37°C. For FGF-2 and aggrecan staining,
deparaffinized sections were first treated with 0.1% hyaluronidase in
acetate buffer, pH 5.0, at 37°C for 30 or 60 minutes, respectively.
For type II collagen staining, deparaffinized sections were pretreated
with 0.1% trypsin (0152–13; DIFCO Laboratories, Detroit, MI) in PBS
for 30 minutes at 37°C. All sections were washed in PBS, and
incubated for 30 minutes in 0.3% H2O2 in
methanol to inactivate endogenous peroxidases. After incubation for 1
hour at room temperature, sections were washed twice in PBS and
incubated with biotinylated anti-rabbit or anti-mouse immunoglobulin
antibody (DAKO Japan Co., Kyoto, Japan). After two additional washes,
they were incubated in peroxidase-conjugated streptavidin (P397; DAKO
Japan Co.) followed by two washes in PBS. Bound peroxidase was
developed with 0.02% 3,3'-diaminobenzidine in 0.1 mol/Tris buffer, pH
7.6, in 0.005% H2O2 for 5 minutes,
and counterstaining was performed with 5% methyl green. As negative
controls, the primary antibodies were replaced with normal mouse or
rabbit serum, PBS, or were preabsorbed with recombinant mature human
ChM-I protein.
The density of CD34 expression was evaluated as follows: -, no signal;
+/-, 1 to 2 capillaries seen in an area of 0.5
mm2
using a micrometer with a x200 objective
magnification; +, 3 to 5 capillaries; ++, 6 to 10 capillaries; +++,
>11 capillaries. For statistical analysis, we used one factor analysis
of variance (analysis of variance) and the chi-square test.
P values <0.05 were considered statistically significant.
|
Results
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ChM-I, FGF-2, Type II Collagen and Aggrecan Expression in Human
Cartilage
Signals for type II collagen were seen throughout the matrix of
the tracheal cartilage and cartilaginous matrix of the vertebral tissue
(Figure 1A)
. Signals for aggrecan were
observed in the matrix of the tracheal cartilage and especially in the
matrix of mature to hypertrophic cartilage of the vertebral tissue
(Figure 1B)
, whereas weak signals were seen throughout the
cartilaginous matrix. Signals for FGF-2 were seen in the proliferating
to mature chondrocytes of the vertebral tissues, and in the
chondrocytes of the tracheal cartilage (Figure 1C)
. Also, signals for
FGF-2 were seen on the osteoclast-like multinucleated giant cells near
the new bone. Strong signals for ChM-I were observed on the
interterritorial regions around the lacunae of the cartilaginous
tissues, especially in the areas around the mature to hypertrophic
chondrocytes, and were also seen throughout the cartilaginous matrix in
the vertebral tissues (Figure 1D)
. Also, signals for ChM-I were seen in
the cytoplasm of the chondrocytes of the tracheal cartilage (data not
shown). However, no signals for ChM-I, type II collagen, or aggrecan
were seen in the normal salivary gland tissue, whereas numerous
CD34-positive capillaries were observed in the stroma of the normal
salivary glands (data not shown).
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Figure 1. Results of control studies for each antibody using endochondral
ossification tissues of the neonatal vertebra. p, proliferating
cartilage; m, mature cartilage; h, hypertrophic cartilage; c,
calcifying cartilage; b, bone. A: Diffuse type II collagen
immunoreactivity in the matrix of the vertebral cartilage. Intense
immunoreactivity was seen around the lacunae of the mature to
hypertrophic cartilage. Weak immunoreactivity was seen around the
proliferating chondrocytes. Although intense signals for type II
collagen were observed on the interterritorial matrix of the calcifying
cartilage, no signals were seen in the bone tissue. B:
Aggrecan immunoreactivity in the matrix of the mature to hypertrophic
cartilage of the vertebral cartilage. Note that diffuse signals for
aggrecan were seen throughout the cartilaginous matrix. C:
Strong FGF-2 immunoreactivity in the proliferating to mature
chondrocytes of the vertebral cartilage. Note that signals for FGF-2
were seen in the cytoplasm of the osteoclasts
(arrowheads).
D: Strong ChM-I immunoreactivity on the interterritorial
matrix around the lacunae of the mature to hypertrophic cartilage of
the vertebral cartilage tissue. Weak ChM-I immunoreactivity was seen
throughout the cartilage matrix. Original magnifications, x100.
|
|
CD34 Expression in Salivary Pleomorphic Adenomas
CD34 is a marker of endothelial cells. The profiles of the 35
cases of pleomorphic adenoma examined in this study are shown in Table 2
. All cases showed typical histological
characteristics. Four stromal types were observed: fibrous (54.3%),
hyaline (40.0%), myxoid (91.4%), and chondroid (68.6%). Fibrous
elements showed spindle-shaped neoplastic myoepithelial cells with
bundles of collagen fibers. Hyaline elements revealed hypocellular
areas with dense eosin staining. Myxoid elements showed a pale
hematoxylin-positive matrix with stellate or spindle-shaped cells.
Chondroid elements showed hyaline cartilage-like areas with lacuna
formation, in which lacuna cells were observed.
Signals for CD34 were seen in only a few areas of pleomorphic adenomas,
ie, capillaries in the fibrous, myxoid and/or hyaline stroma, and the
areas around epithelial components, but CD34 expression was never
observed in the chondroid component (P < 0.001)
(Figure 2, A–C)
. We evaluated the
expression and density of CD34-positive capillaries (Table 2)
.
CD34-positive capillaries were frequently seen in the fibrous stroma,
but they were infrequently observed in the hyaline and myxoid stromas
(P < 0.05). CD34 positivity was significantly
different between the chondroid stroma and other stromas. On the other
hand, pleomorphic adenomas showed numerous CD34-positive capillaries in
the fibrous capsules (Figure 2D)
.
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Figure 2. A: A few CD34-positive capillaries were seen
in the fibrous area of pleomorphic adenoma. B: Few
CD34-positive capillaries were seen in the myxoid area of pleomorphic
adenoma. Scale bar, 120 µm. C: No immunoreactivity for
CD34 was seen in the chondroid area of pleomorphic adenoma. Scale bar,
120 µm. D: CD34 immunoreactivity was seen in the fibrous
capsules of pleomorphic adenoma. f, fibrous capsule; t, tumor; n,
normal salivary gland. Note that many CD34-immunoreactive capillaries
were seen in the normal salivary gland. Original magnifications,
x120.
|
|
FGF-2 and ChM-I Expression in the Salivary Pleomorphic Adenomas
Signals for FGF-2 were seen in the lacuna cells in the chondroid
elements and also on the basement membrane regions of the solid nests
of the neoplastic myoepithelial cells or around the spindle-shaped or
stellate cells in the myxoid elements (Figure 3A)
. These results were compatible with
those of our previous study.3
FGF-2 positivity, however,
was not significantly different between myxoid/chondroid stromas and
other stromas (Table 3)
.
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Figure 3. A: Strong FGF-2 immunoreactivity was seen in the lacuna
cells of the chondroid area of pleomorphic adenoma. Note that FGF-2
immunoreactivity was seen in the pericellular spaces of the lacuna
cells. B: Diffuse ChM-I immunoreactivity was seen in the
interterritorial spaces around lacunae of the chondroid area of
pleomorphic adenoma. Weak signals were seen throughout the chondroid
matrix. Inset: Strong immunoreactivity for ChM-I was seen in
the interterritorial regions of the lacuna cells. C: No
immunoreactivity for ChM-I was seen after pre-absorption of the
antibody with recombinant human ChM-I protein. c, chondroid area. Scale
bar, 100 µm. D: Moderate ChM-I immunoreactivity was seen
in the spindle-shaped neoplastic myoepithelial cells of myxoid area of
pleomorphic adenoma. Note that weak immunoreactivity was seen in the
myxoid matrix of pleomorphic adenoma. Original magnifications: x200
(A and
D), x100
(B and
C), and x256
(inset in
B).
|
|
Strong signals for ChM-I were observed on the interterritorial spaces
around the lacunae, whereas weak signals were seen in the chondroid
matrix (Figure 3B
and Table 4
). Immunoreactivity for ChM-I was observed
in the neoplastic myoepithelial cells of the myxoid elements (Figure 3D)
. In the chondroid areas, FGF-2 was immunolocalized to the surface
and cytoplasm of the lacuna cells, whereas ChM-I was immunolocalized to
the interterritorial spaces around the lacunae. Pre-absorption of the
antibody with recombinant human ChM-I protein completely abolished
signals for ChM-I, and no staining was seen in negative controls
(Figure 3C)
.
Type II Collagen and Aggrecan Expression in the Salivary
Pleomorphic Adenomas
Strong signals for type II collagen were seen throughout the
chondroid matrix, but none were seen in other mesenchymal-like elements
or epithelial elements (Figure 4A)
. On the
other hand, strong signals for aggrecan were observed consistently in
the matrix of the chondroid elements (Figure 4B)
. Such signals were
also seen in small myxoid areas within or near the epithelial sheets,
and occasionally in the intercellular spaces of the solid nests of the
neoplastic myoepithelial cells (Figure 4, C and D)
. These results were
similar to those reported previously.8
Aggrecan positivity
was significantly different between myxoid/chondroid stromas and other
stromas (P < 0.001) (Tables 3 and 4)
. No
staining was observed in negative controls.
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Figure 4. A: Diffuse type II collagen immunoreactivity was seen in the
chondroid matrix of pleomorphic adenoma. B: Diffuse aggrecan
immunoreactivity was seen in the chondroid matrix of pleomorphic
adenoma. C: Diffuse aggrecan immunoreactivity was seen in
the myxoid area. D: Moderate aggrecan immunoreactivity was
seen in the interspaces among the tubulo-glandular structures.
Asterisks, tubulo-glandular structures. Scale bars, 100
µm. Original magnifications, x100.
|
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Discussion
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Pleomorphic adenoma of the salivary glands, the most frequent type
of benign tumor, is considered an epithelial tumor. However,
pleomorphic adenoma shows various histological features, including
myxoid and chondroid characteristics.9,10
We reported
previously that chondroid areas of pleomorphic adenoma expressed type
II collagen protein and mRNA, and lacuna cells were similar to
authentic chondrocytes in phenotype.1
The present study
also showed the deposition of type II collagen and aggrecan in the
stroma of the chondroid areas. Aggrecan is a cartilage-specific large
aggregating chondroitin sulfate proteoglycan, and our results were
similar to those reported previously,8
ie, aggrecan was
deposited in the chondroid matrix of pleomorphic adenoma. The BMP-2
produced by neoplastic myoepithelial cells may induce chondroid tissue
by a paracrine mechanism,1
and the BMP-6 produced by
lacuna cells may maintain chondroid tissue by an autocrine mechanism in
pleomorphic adenoma.2
Thus, the co-expression of FGF-2 and
FGF receptor-1 may inhibit osteogenesis in the chondroid
areas.3
BMP-2 and FGF-2 are expressed in neoplastic
myoepithelial cells of myxoid areas of pleomorphic adenomas, which may
be related to the differentiation of neoplastic myoepithelial cells and
mesenchymal-like tissue formation including fibrous, hyaline, myxoid,
and chondroid tissues.
On the other hand, although FGF-2 is a strong angiogenic
factor,11-13
pleomorphic adenoma is a hypovascular tumor
as indicated by the CD34 expression and density of endothelial cells in
this type of tumor. We expected there to be some strong angioinhibitors
in pleomorphic adenoma, and a good candidate was ChM-I, a 25-kd
glycoprotein purified from bovine epiphyseal cartilage,4
that was recently identified as a novel endothelial cell growth
inhibitor.5,6
Cartilage is generally avascular and
exhibits resistance to vascular invasion because of an intrinsic
angiogenesis inhibition, which is because of the effects of ChM-I.
ChM-I was reported to inhibit DNA synthesis and tube morphogenesis of
cultured vascular endothelial cells in vitro,14
but this molecule stimulated DNA synthesis and proteoglycan synthesis
in cultured growth plate chondrocytes in
vitro.4,12
ChM-I is a glycoprotein of 121 amino acid
residues and is encoded as the C-terminal portion of a large precursor
(335 amino acids).4
Mature ChM-I is assumed to be secreted
from chondrocytes after proteolytic cleavage by a
precursor-endoprotease, furin.15,16
In situ
hybridization analysis indicated that ChM-I mRNA was expressed in the
proliferating to upper hypertrophic chondrocytes in the growth plate of
the long bones, but the expression of the gene was reduced in the lower
hypertrophic and calcified zone, allowing vascular
invasion.6
Immunohistochemical examination clearly
indicated that the localization of ChM-I protein completely overlapped
the area of its gene expression reported previously.6
ChM-I was accumulated in the interterritorial spaces around the lacunae
of the cartilage matrix as well as on the cartilage matrix
itself. In contrast, FGF-2 protein was confined to the cell surface or
pericellular space in the territorial regions. Thus, the distribution
of ChM-I protein in cartilage was clearly distinct from that of FGF-2.
Immunostained FGF-2 is tightly associated with the cell surface,
whereas ChM-I diffuses away from the cells, explaining the avascular
nature of the tumor in the chondroid areas despite the presence of both
angiogenic and anti-angiogenic factors. The unique localization pattern
may explain how ChM-I exerts its anti-angiogenic effect on cartilage.
Recently, we reported that BMP-6 was expressed in the lacuna
cells of the chondroid areas in pleomorphic adenoma.2
In
the present study, we also demonstrated that aggrecan was deposited in
the chondroid matrix in this type of tumor. The phenotype
of lacuna cells in chondroid areas was similar to that of mature to
upper hypertrophic chondrocytes with regard to expression of aggrecan
and BMP-6.2,17
On the other hand, FGF-2 was expressed in
the proliferating to mature chondrocytes of the areas of
endochondral ossification, whereas the lacuna cells in chondroid
areas showed strong expression of FGF-2. Our results showed that,
similarly to aggrecan, ChM-I was localized to the
interterritorial spaces around the lacunae in the chondroid matrix,
which is an avascular region in pleomorphic adenoma. These findings
suggested that the phenotype of chondroid areas of pleomorphic adenoma
was an authentic mixed cartilaginous phenotype, but the phenotype
of the lacuna cells in chondroid areas may be similar to that of
the mature to upper hypertrophic chondrocytes. Type X collagen is
expressed in the lower hypertrophic chondrocytes and therefore the
expression of this molecule in pleomorphic adenomas is presently under
investigation in our laboratory.
In conclusion, pleomorphic adenomas of the salivary glands
expressed ChM-I protein in the chondroid areas, and ChM-I plays an
important role in determining the avascular features of the chondroid
area of this type of tumor, although the ubiquitous angiogenic growth
factor FGF-2 was localized to lacuna cells of chondroid areas. Thus,
with regard to expression of ChM-I protein, pleomorphic adenoma should
be classified as a hypovascular tumor.
|
Acknowledgements
|
|---|
We thank S. Skiguchi, K. Wakamatsu, S. Hashimoto, A.
Niizeki, and Y. Aoki; the staff of the Department of Pathology,
Japanese Red Cross Medical Center, Tokyo, Japan, for their technical
assistance; Mr. Takashi Yoshizawa, the Photo Center, Tokyo Medical and
Dental University, Tokyo, Japan, for microphotography; and Drs.
Toshikazu Yada and Koji Kimata, Institute for Molecular Science of
Medicine, Aichi Medical University, Nagoya, Japan, for generously
providing specific antibody against aggrecan.
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Footnotes
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Address reprint requests to Kimihide Kusafuka D.D.S., Ph.D, Department of Pathology, Japanese Red Cross Medical Center, 4-1-22 Hiroo, Shibuya-ku, Tokyo 150-8935, Japan.
Accepted for publication January 10, 2001.
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References
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Top
Abstract
Introduction
