© 1995 American Heart Association, Inc.
Articles |
Genetic Determination of Cartilaginous Metaplasia in Mouse Aorta
From the Department of Medicine (J.-H.Q., L.L.D., A.J.L.) and the Department of Microbiology and Molecular Genetics and Molecular Biology Institute (J.-H.Q., A.J.L.), University of California, Los Angeles, the Department of Pathology, Cedars-Sinai Medical Center (M.C.F.), and the Department of Physiology (L.L.D.), University of California, Los Angeles.
Correspondence to Jian-Hua Qiao, MD, Division of Cardiology, Department of Medicine, 47-123 CHS, University of California, Los Angeles, 10833 Le Conte Ave, Los Angeles, CA 90024-1679.
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Abstract Calcification frequently occurs in atherosclerotic plaques in humans, but the cellular and genetic factors contributing to this pathological trait are unknown. We previously reported that the arterial calcification among inbred strains is genetically determined, and we now report that cartilaginous metaplasia, associated with the presence of arterial chondrocytes that express type II collagen, may underlie this calcification. Both uncalcified and calcified cartilaginous metaplasia were often colocalized with aortic atheromatous lesions and calcification, and clear genetic differences were observed in the occurrence of aortic cartilaginous metaplasia among inbred strains. Analysis of a genetic cross between strains C57BL/6J (exhibiting aortic cartilaginous metaplasia) and C3H/HeJ (no aortic cartilaginous metaplasia) revealed a recessive inheritance pattern; thus, F1 mice were entirely devoid of cartilaginous metaplasia, in common with the C3H/HeJ parental strain. Analyses of an F2 cross and a set of recombinant inbred strains derived from parental strains C57BL/6J and C3H/HeJ were consistent with a major gene effect exhibiting incomplete penetrance. The occurrence of aortic calcification was correlated with the occurrence of cartilaginous metaplasia in these genetic crosses, suggesting a link between the traits. Finally, we observed widespread calcified cartilaginous metaplasia within spontaneous atherosclerotic lesions in mice targeted for a null mutation in the apoE gene, suggesting that cartilaginous metaplasia is a potential pathway for artery wall calcification associated with the atherosclerotic plaque.
Key Words: atherosclerosis • calcification • genetics • mouse strains, inbred • artery wall
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Introduction |
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Arterial calcification has been associated with occlusive coronary artery disease in human populations1 2 3 4 5 6 7 8 9 10 and may contribute to plaque rupture and myocardial infarction.1 5 The presence of coronary artery calcification as detected by fluoroscopy has been shown to have prognostic value in selected populations.6 Calcification is also a major complication limiting the longevity of bioprosthetic valves.11 12 13 Calcification is a complex process that occurs in normal biological processes (eg, the calcium mineral deposition of bone and dentin) and numerous pathological conditions involving irreversible degeneration or cell death (eg, advanced atherosclerotic plaque, tuberculosis, and neoplasms). The factors contributing to arterial calcification are unknown, but the fact that some plaques exhibit considerable calcification whereas others are free of calcification suggests that either genetic or environmental factors are likely to be of importance.
We have reported that arterial calcification is influenced by both genetic and dietary factors in a mouse model.14 Aortic calcification is often accompanied by the presence of cartilaginous metaplasia. In the present study, we observed a clear genetic difference in the occurrence of aortic cartilaginous metaplasia among inbred strains. The cartilaginous metaplasia was frequently associated with arterial calcification, and in one set of RI strains the two traits tended to cosegregate. The calcified cartilaginous metaplasia was also present in the atherosclerotic plaques in apoE gene–targeted (knockout) mice, which exhibit severe spontaneous arterial atherosclerosis and calcification when fed a regular chow diet.14 15 16 17 18 These findings suggest a potential relation among atherosclerosis, cartilaginous metaplasia, and calcification.
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Methods |
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Mice and Diets
Inbred strains of mice were obtained from the Jackson Laboratory (Bar Harbor, Me), except the C57BL/10ScSnA and C3H/DiSnA strains (from Dr Peter Demant, Netherlands Cancer Institute, Amsterdam, Netherlands), apoE knockout mice (a gift from Dr N. Maeda, University of North Carolina, Chapel Hill),15 and MHC class II–deficient mice (purchased from GenPharm International, Mountain View, Ca). BxH F2 mice were bred in our laboratory. The mice were fed either standard rodent chow containing 4% fat (Purina 5001) or a high-fat, high-cholesterol (atherogenic) diet (TD 90221, Food-Tek, Inc) containing 15% (wt/wt) fat, 1.25% cholesterol, and 0.5% cholic acid.14 Animals were housed 3 to 5 per cage and maintained in a temperature-controlled room with a 12-hour light/dark cycle. All mice were females between 4 and 6 months of age at the time of euthanasia, except for the atherogenic diet–fed group of DBA/2J mice, which were older than 10 months of age, and chow-fed groups of BxH F1 and apoE knockout mice, which comprised both males and females.
Histopathologic and Immunohistochemical Studies
Animals were killed by cervical dislocation after isofluorane
(Forane, Anaquest) anesthesia. The heart and proximal aorta
(including the aortic arch) were excised and washed in
phosphate-buffered saline. The basal portion of the heart and the
root of the aorta were embedded in OCT compound (Tissue-Tek) and frozen
on dry ice. Serial 10-µm-thick cryosections (every fifth section
from the lower portion of the ventricles to the appearance of aortic
valves, every other section in the region of the aortic sinus, and
every fifth section from the disappearance of the aortic valves to the
aortic arch) were collected on poly-D-lysine coated slides
and stored at -70°C until histological staining was
done.14
Sections were stained with oil red O and hematoxylin and counterstained with fast green for the identification of atheromatous lesions (fatty streaks), arterial calcification, and aortic cartilaginous metaplasia.14 19 20 21 For confirmation of calcium mineral deposits, representative sections were also stained by the alizarin red S and von Kossa techniques.14 Sections of mouse trachea and femoral bone served as positive controls for cartilage (chondrocytes) and calcification. Every stained section was examined by light microscopy for the presence of cartilage and calcium deposits in the arterial wall. Cartilaginous metaplasia was defined as the presence of chondrocytes in lacunae within a collagenous mucopolysaccharide–rich matrix in aortic wall or valve attachments. Mice were considered positive if cartilage and/or calcium deposits were observed in the aortic wall in one or more sections.
For immunochemical identification of the chondrocytes present in the aorta, we applied rabbit anti-human collagen type II polyclonal antiserum (Chemicon International Inc), which cross-reacted with chondrocytes of mouse tracheal wall, and the avidin–biotinylated peroxidase system to stain frozen mouse aortic sections that contained loci of cartilaginous metaplasia. The final working dilution of this antibody was 1:200, and omission of primary antibody or the use of other nonrelevant rabbit antiserum was performed as a negative control. The immunohistochemical staining procedures were previously described in detail.14 20
Statistical Analysis
Data analysis was performed using STATVIEW
(Student's t test, 
2
analysis, and ANOVA) software for the Macintosh personal
computer.
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Results |
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Aortic Cartilaginous Metaplasia
During the course of studies of fatty streak formation in mouse aorta, we observed areas of cartilage-like tissue in the proximal aorta of certain inbred strains that closely resembled cartilage in the tracheal wall (see inset, Fig 1A
).
Immunohistochemical staining confirmed that the tissue had undergone
cartilaginous metaplasia. Type II collagen, the major matrix protein of
cartilage cells, is a marker for cartilage. A polyclonal antibody,
prepared in rabbit anti-human type II collagen, was found to
cross-react well with mouse type II collagen as judged by strong
positive reaction with mouse tracheal wall chondrocytes (Fig 2B). Very similar immunoreactivity of this antibody was
observed in areas of the aorta containing the cartilage-like tissue
(Fig 2A). Calcium-mineral deposition was confirmed by histochemical
staining using the alizarin red S and von Kossa staining techniques
(see inset, Figs 2A as well as Fig 3B and 3C), with
mouse femoral bone tissue serving as a positive control.
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Cartilaginous metaplasia occurred most often within the aortic
ring and valve commissure attachment areas (proximal aorta) (Fig 1A
through 1C). Both uncalcified and calcified forms of cartilaginous
metaplasia were observed in the normal aorta (mice fed chow) and the
aorta with atheromatous lesions (mice fed the
high-fat diet) (Fig 1). No definite cartilaginous metaplasia was
observed in the coronary arteries and cardiac valves. The
distal thoracic and abdominal aorta were not examined in this study.
Loci of uncalcified cartilaginous metaplasia frequently exhibited
positive staining with the lipophilic dye oil red O (Fig 1A through 1C
and 1F), which is a typical histological feature of
hyaline cartilage. We frequently observed typical arterial
calcification in sections adjacent to those containing calcified
cartilaginous metaplasia (Fig 1E and 1F). For example, among C57BL/6J
mice fed the atherogenic diet that stained positively (and that
exhibited calcified cartilaginous metaplasia), 94% (15 of 16) also had
typical aortic calcification in the adjacent sections. The
characteristics of histological staining for
uncalcified cartilaginous metaplasia, calcified cartilaginous
metaplasia, and typical calcification are shown in Table 1.
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Genetic Control of Cartilaginous Metaplasia
Inbred strains of mice differed in the occurrence of aortic
cartilaginous metaplasia. For example, on a low-fat–chow diet,
aortic cartilaginous metaplasia was observed in about 23% of strain
C57BL/6J mice, whereas no aortic cartilage was observed in several
other common inbred mouse strains, such as BALB/cJ, A/J, and C3H/HeJ
(Table 2). Two MRL/MPJ substrains exhibited a very high
frequency of aortic cartilaginous metaplasia (57% and 89%). Feeding
the mice an atherogenic diet for 15 weeks did not significantly
influence the occurrence of aortic cartilaginous metaplasia in most of
the inbred strains (Table 2).
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Differences were present between C57BL/6J and MRL/MPJ substrains in
terms of the ratio of subtypes of aortic cartilaginous metaplasia
(calcified versus uncalcified). For example, none of the aortic
cartilage (0 of 25, 0%) in MRL/MPJ substrains fed either chow or an
atherogenic diet was calcified. However, nearly one half of the aortic
cartilage (4 of 9, 44%) in strain C57BL/6J mice fed chow was
calcified. Interestingly, the atherogenic diet appeared to increase the
occurrence of calcified aortic cartilage in this strain. Thus, 4 of 39
(10%) of mice in the chow-fed group and 16 of 60 (27%) in the
atherogenic diet–fed group of C57BL/6J mice exhibited calcified
cartilaginous metaplasia (2=3.95,
P<.05).
To evaluate whether immunological factors contribute to the process of
aortic cartilaginous metaplasia, we examined two strains of mice that
lack an intact immune system. The occurrence of aortic cartilaginous
metaplasia in these mice is shown in Table 2 and Fig 3A through 3C.
Nude mice22 ; class II MHC antigen–deficient
mice,23 which lack CD4+ T helper lymphocytes; and
op/op mice24 all had the ability to produce the
aortic cartilaginous metaplasia.
Inheritance of Aortic Cartilaginous Metaplasia
Strain C57BL/6J mice exhibited a relatively high occurrence of
aortic cartilaginous metaplasia on both chow and atherogenic diets,
whereas strain C3H/HeJ mice exhibited no evidence of this trait. The
differences between the two strains were significant for both mice fed
chow and those fed a high-fat diet. To further examine the role of
genetics in development of aortic cartilaginous metaplasia, we
characterized F1 and F2 progeny as well as a
set of BxH RI strains for the occurrence of aortic cartilaginous
metaplasia (Tables 3 and 4).
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Aortic cartilaginous metaplasia was absent in all BxH F1
progeny in common with the C3H/HeJ parent, indicating that this trait
exhibits recessive inheritance. Seven percent of the BxH
F2 progeny exhibited aortic cartilaginous metaplasia. This
is not significantly different from the 8% predicted for a single,
recessive mendelian gene with 33% penetrance (33% is the average
occurrence in strain C57BL/6J mice fed either chow or a high-fat
diet, Table 3). Among positive F2 progeny that exhibited
aortic cartilaginous metaplasia, four (4 of 13, 31%) were
calcified.
We further examined the inheritance of aortic cartilaginous metaplasia
in a set of BxH RI strains. Each member of the BxH RI strains
contains a unique mixture of genes derived from the parental strains.
These recombinant genotypes have been fixed by many generations
of inbreeding.25 26 These strains have been previously
typed for aortic calcification and aortic
atherogenesis.14 21 The distribution of aortic
cartilaginous metaplasia among the RI strains is presented in
Table 4. The majority of the BxH RI strains (7 of 10) exhibited no
aortic cartilaginous metaplasia, resembling the phenotype of
the C3H/HeJ parent (0%), whereas 3 of 10 strains developed this trait
(Table 4). If cartilaginous metaplasia was determined by a single major
gene, it would be expected to occur in 
50% of the RI strains; this
is not significantly different from the observed value. These patterns
of inheritance are consistent with (but do not prove) the
hypothesis that the aortic cartilaginous metaplasia is determined by a
single major gene exhibiting incomplete penetrance.
The occurrence of aortic cartilaginous metaplasia was significantly
correlated with calcification in the aortic root among the BxH RI
strains (r=.76, P=.01) (Table 4). Clearly,
however, there are additional factors contributing to calcification.
For example, C3H/HeJ and several other common laboratory inbred strains
exhibited no evidence of arterial cartilaginous metaplasia,
yet they developed arterial calcification in aorta and
coronary arteries.14 Given the likely complexity
of calcification, multiple mechanisms may be involved.
Cartilaginous Metaplasia in ApoE Knockout Mice
Using gene targeting techniques, apoE knockout mice recently were
created.15 16 These genetically manipulated mice develop
severe hypercholesterolemia due to delayed
clearance of large atherogenic particles from the circulation and
exhibit the entire spectrum of lesions observed during human
atherogenesis, from fatty streaks to complex lesions.14 15 16 17 18
One feature of the apoE knockout mice is enhanced artery wall
calcification compared with most laboratory-inbred
strains.14 We examined 6 apoE knockout homozygotes of a
mixed genetic background derived from mouse strains 129/J and C57BL/6J.
All these mice (6 of 6, 100%) developed extensive spontaneous
atherosclerosis at 4 to 6 months old, and 4 (4 of 6,
67%) exhibited typical calcification in aortic
atheromatous lesions. Calcified cartilaginous
metaplasia was found in 3 of 6 apoE knockout mice (50%). Unlike strain
C57BL/6J mice, in which most of the cartilaginous metaplasia was
located in aortic ring or aortic valve attachments (32 of 33, 97%),
apoE knockout mice exhibited cartilaginous metaplasia within the
atherosclerotic plaques in the aortic arch (3 of 3, 100%) (Fig 3D and 3E) and pulmonary artery (1 of 3, 33%) (Fig 3F). Thus, either
hypercholesterolemia or the consequent
atherosclerosis appears to promote cartilaginous
metaplasia. It is noteworthy that in strain C57BL/6J mice, both
atherosclerosis and cartilaginous metaplasia are
largely restricted to the proximal aorta, whereas in apoE knockout
mice, both traits are more widely dispersed.
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Discussion |
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Our results indicate a genetic component contributing to aortic cartilaginous metaplasia and provide presumptive evidence that cartilaginous metaplasia is one of the pathways contributing to arterial calcification. The finding that different inbred strains of mice show striking differences in cartilaginous metaplasia despite being maintained under very similar environmental conditions indicates the existence of hereditary factors contributing to the trait. This possibility is supported by the inheritance patterns observed in crosses between strain C57BL/6J (susceptible) and strain C3H/HeJ (resistant) mice. The explanation for the incomplete penetrance of the trait is unclear. It may be related to subtle environmental or developmental influences or it could be due to stochastic or sampling factors. Results of both the F2 cross and the RI strain studies are consistent with the possibility that a major gene underlies the development of cartilaginous metaplasia, but additional studies are required to demonstrate this.
A significant correlation between the occurrence of cartilaginous metaplasia and aortic calcification was observed among the BxH RI strains (r=.76, P=.01). Moreover, calcification was frequently observed adjacent the sites of cartilaginous metaplasia in histological studies. These findings support the possibility that cartilaginous metaplasia may be one mechanism contributing to arterial calcification. In addition, studies of genetically engineered mice that exhibit extreme hypercholesterolemia because of a null mutation of the apoE gene (apoE knockout mice) suggest a link among cartilaginous metaplasia, calcification, and atherogenesis. Thus, in contrast to normal inbred strains of mice, apoE knockout mice exhibit large and widely dispersed atherosclerotic lesions throughout the aorta.14 15 16 17 18 Whereas cartilaginous metaplasia and arterial calcification in normal mice were restricted to the proximal aorta, apoE knockout mice exhibited both in the aortic arch.
The fact that arterial calcification occurs in some strains in the absence of detectable cartilaginous metaplasia suggests that there may be different pathways leading to calcification or that the rate of progression to calcification differs between strains. Indeed, the fact that calcified cartilaginous metaplasia was present in strain C57BL/6J but not in MRL/MPJ substrains suggests that genetic factors also affect the calcification of cartilagenous metaplasia. There are a number of bone-associated proteins and regulatory factors that may contribute to the observed genetic differences in cartilaginous metaplasia and calcification. Our strategy is first to map the genes responsible and then to focus on any genes that reside in the chromosomal region identified.
Cartilaginous metaplasia in cardiovascular tissues has been reported in mice, rats, rabbits, and other animals, as well as humans.27 28 29 30 31 In most instances the cartilage is hyaline, but in sheep the cartilage may change to bone through enchondrial ossification.28 The mechanisms responsible for arterial wall cartilaginous metaplasia are unknown. Aortic wall hyaline cartilage was induced in chickens by intramural injection with carrageenan.32 It has been reported that mechanical stress (combined compression and rotation) induced metaplastic changes from typical elongated fibrous tissue cells to typical rounded cartilage cells.33 Our observation that cartilaginous metaplasia is localized mainly in the aortic valve attachments, the region that faces strong mechanical stress during the cardiac cycle, is consistent with the hypothesis that mechanical stress is one factor promoting cartilaginous metaplasia in the mouse aortic wall. However, the significant differences in the occurrence of cartilaginous metaplasia among several common inbred strains of mice suggest that genetic factors also play an important role. Although the mechanism of conversion of hyaline (uncalcified) cartilage to calcified cartilage is unclear, experiments in avian embryos demonstrate that the constitutive expression of the v-myc oncogene maintains chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.34 In vitro data also suggest that c-fos oncogene may play a crucial role in the osteogenic differentiation of cartilage.35
It is unknown whether the immune system and inflammatory mechanisms are involved in physiological and pathological calcification. It has been reported that experimental calcification of porcine bioprosthetic xenograft tissue does not require normal T-lymphocyte activity in nude mice.36 The finding of calcification of rat aorta in a grafted intraperitoneal (0.22-µm) millipore chamber suggests that calcification can occur in the absence of scavenging by inflammatory cells such as macrophages.37 Our results in nude and op/op mice also support the notion that normal T lymphocytes and macrophages are not essential for hyaline or calcified arterial wall cartilaginous metaplasia.
Although calcification is a prominent feature of atherosclerotic lesions, little is known about the biology of arterial wall calcification. Traditionally, calcification has been considered to be an end-stage degenerative process associated with complex atherosclerosis. However, recent observations of expression of bone-associated proteins in atherosclerotic lesions suggest that human arterial calcification is an active, regulated process.38 39 40 41 42 43 44 45 A combination of in situ hybridization and immunohistochemical techniques has revealed that smooth muscle cells as well as macrophages in human atheromatous plaques can express osteopontin mRNA and protein.40 41 42 43 Extracellular osteopontin protein is often found in areas of dense connective tissue that is colocalized with calcification in the plaque.43 44 Results of other experiments have suggested the presence of a unique subpopulation of artery wall cells, called calcifying vascular cells, in the wall of human and bovine arteries.39 45 Although cartilaginous metaplasia has not been identified in human atherosclerotic lesions, our studies of aortic cartilaginous metaplasia and calcification in mice suggest that some vessel wall cell populations can differentiate to chondrocytes, form cartilage, and contribute to calcification. This hypothesis was supported by our finding that the relatively advanced atherosclerotic lesions present in apoE knockout mice were associated with both cartilaginous metaplasia and calcification. Thus, cartilaginous metaplasia is a potential pathway of artery wall calcification in the atherosclerotic plaque. The study of genetic factors contributing to arterial cartilaginous metaplasia and calcification may provide an understanding of the molecular and cellular mechanisms involved in these processes.
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Acknowledgments |
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This work was supported in part by National Institutes of Health award HL-30568 and the Laubisch Fund, University of California, Los Angeles. We wish to thank X.-P. Wang and P.-Z. Xie for their excellent histological technical assistance, F. Liao for the maintenance of strain BxH genetically crossed mice, A. Fyfe for the maintenance of immune-deficient mice, S. Shoemaker for the maintenance of op/op mice, and S. Zhang and N. Maeda (University of North Carolina) for providing us with mice lacking apoE.
Received November 3, 1994; accepted May 24, 1995.
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