© 1995 American Heart Association, Inc.
Articles |
Alterations of Bone Matrix Protein mRNA Expression in Rat Aorta In Vitro
From the Department of Pathology, National Cardiovascular Center, and the Department of Pathology, Osaka University Medical School, Suita, Japan.
Correspondence to Hiroyuki Hao, MD, Department of Internal Medicine II, Nihon University Surugadai Hospital, Kandasurugadai 1-8-13, Chiyoda-ku, Tokyo 101, Japan.
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Abstract |
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Abstract We examined the expression of matrix Gla protein (MGP), osteopontin (OPN), and osteonectin (ON) mRNAs in aortic rings excised from 3-month-, 10-month-, and 2-week-old rats during 72-hour incubations in serum-free media. In the aortic rings from 3-month-old rats, the expression of MGP mRNA was strong before incubation and increased during the 72-hour incubation. The expression of OPN mRNA was first detected after a 5-hour incubation and increased thereafter, and that of ON mRNA was strong before the incubation and decreased during the incubation. The expression of MGP and OPN mRNAs in 10-month- and 2-week-old rats was similar to that in 3-month-old rats. In contrast, expression of ON mRNA in 10-month-old rats and the expression of ON mRNA in 2-week-old rats was stronger than that in 3-month-old rats at every incubation period. In situ hybridization and immunohistochemistry identified the MGP, OPN, and ON mRNA–expressing cells as vascular smooth muscle cells. These results suggest that the expression of these mRNAs was regulated in incubation time–dependent and age-specific ways. We believe that this organ culture model is useful for further studies of the function of these bone matrix proteins and regulation of their expression in the vessel wall.
Key Words: matrix Gla protein • smooth muscle cell • osteopontin • osteonectin • gene expression
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Introduction |
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The structural basis of aortic aging, which can be regarded as an inevitable physiological process, is most conspicuous in the intima but also involves the media.1 Throughout life, the aortic wall usually thickens by the progressive accumulation of connective tissue matrix such as collagen, proteoglycans, elastin, and a number of other ECM glycoproteins.1 It is generally accepted that interactions between ECM proteins and other proteins can influence cellular shape, motility, and growth in vascular complications such as atherosclerosis.2 3 Therefore, both qualitative and quantitative alterations in the ECM proteins might play a key role in the development of vascular complications. Although MGP, OPN, and ON are representative bone-related noncollagenous ECM proteins, there are some reports that the mRNAs for these proteins are expressed by VSMCs.4 5 6 7 8
MGP is a low-molecular-weight (14 Kd),
-carboxyglutamic acid–containing, vitamin K–dependent
protein.9 10 Treatment of rats with warfarin results in
excessive mineralization of bone and cartilage, suggesting that MGP
plays a role as an inhibitor of
mineralization.11 OPN is a 44-Kd
phosphorylated glycoprotein; its amino acid
sequence, RGD, elicits binding of integrin.12 OPN shows a
high affinity for binding to hydroxyapatite13 and appears
to play a role in modulating mineralization of calcifying
tissues.14 ON is a 38-Kd
phosphorylated glycoprotein that shows high
affinity for calcium, hydroxyapatite, and type I collagen. ON is also
considered to play a role in modulating the mineralization of
calcifying tissues.15 16 However, the precise function of
these three ECM proteins remains to be clarified, especially in
vascular systems.
A procedure for measuring both protein biosynthesis and mRNA transcription using rat aortic rings is available17 18 in which in vitro changes appear to reflect the in vivo changes that occur in vascular injury and repair.18 As OPN mRNA is overexpressed by VSMCs in vascular injury and repair,6 we hypothesized that the incubation time in the procedure would influence the production of bone matrix proteins. In the present study, we examined whether incubation time–dependent changes were observed in the expression of MGP, OPN, and ON mRNAs in aortic rings from 3-month-old rats during a 72-hour incubation. We used aortic rings from 10-month- and 2-week-old rats to examine whether age-specific changes were observed in the expression of those mRNAs. The expression of the mRNAs depended on both the incubation time of the aortic rings and the aging of rats.
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Methods |
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Preparation of Aortic Rings
For in vitro culture of aortic rings, 3-month-old (n=60), 10-month-old (n=60), and 2-week-old (n=90) male Wistar rats were purchased from Shizuoka Laboratory Animal Center. They were killed under ether anesthesia.17 18 The region of the aorta from the arch to the diaphragm was used, taking off the branches at the root. Aortas were removed carefully to avoid stretching or compression of the tissue and cleaned of periadventitial tissue. In 3- and 10-month-old rats, 6 or 7 rings (
5 mm long) were obtained from
each aorta; in 2-week-old rats, only 4 or 5 rings (5 mm long) were
obtained. Sixteen rings were randomly placed in each tube with 16 mL
serum-free Dulbecco's modified Eagle's medium supplemented with
penicillin and streptomycin. To optimize nutrition of the aortic rings,
the tubes were placed on a seesaw-type shaker (30 cycles/min) at 37°C
in an atmosphere of 95% air and 5% CO2. The rings were
harvested at 0, 1, 3, 5, 12, 24, 48, and 72 hours. Tissue viability
after a 72-hour incubation in serum-free media has been demonstrated by
Hosoi et al.18 Three rings were used for
histological examination, and the other 13 rings were
used individually for RNA extraction. Aortic rings for in situ
hybridization and immunohistochemistry were fixed with 4%
paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.0)
overnight and embedded in paraffin. Serial sections were cut 5 µm
thick and mounted on 3-aminopropyltriethoxysilane–coated slides.
Probe Preparation
A 1.0-kb fragment of mouse ON cDNA and a 1.2-kb fragment of
mouse OPN cDNA14 were used as hybridization probes. The
specificity of these mouse cDNAs for the detection of rat mRNAs has
been examined.19 A 0.48-kb fragment of rat MGP cDNA (1
through 476)10 was obtained by reverse transcription of
mRNA from newborn rat bone tissue, subjected to polymerase chain
reaction, and subcloned into pBluescript KS. Sequencing of the
resulting cDNA was performed by using the method of Sanger et
al20 ; the base sequence was identical to that of rat MGP
cDNA.10 For in situ hybridization, the shorter fragments
of OPN and ON cDNAs were prepared to facilitate penetration into the
tissue. We compared the specificity and magnitude of the signals
between long and short probes, and the results were not significantly
different.
RNA Extraction and Northern Blotting
Total RNA was extracted from the aortic rings by using the
method of Chirgwin et al.21 For Northern blotting, 10 µg
total RNA was fractionated on a 1% agarose gel and transferred to a
Hybond-N+ nylon membrane (Amersham). The membrane was prehybridized and
then hybridized at 50°C with the [32P]dCTP-labeled
probe according to the manufacturer's instructions. After
hybridization, the membrane was washed at 50°C in 0.1x standard
saline citrate and 0.1% sodium dodecyl sulfate, and signals
were detected by autoradiography. The membrane was
repeatedly rehybridized with other probes. Equal loading was confirmed
both by the staining of 28S and 18S RNA bands with ethidium bromide and
by the rehybridization with ß-actin probe. The resulting
autoradiograms were quantified by scanning laser
densitometry (LKB 2400 GelScan XL) and normalized with both the density
of a ß-actin autoradiogram and the internal control
by blotting mRNA of 3-month-old rats together with mRNAs of 10-month-
or 2-week-old rats.
In Situ Hybridization
Details of the in situ hybridization techniques are
available.22 Digoxigenin-labeled single-strand RNA probes
were prepared for hybridization by using a DIG RNA labeling kit
(Boehringer Mannheim Biochemica) according to the
manufacturer's instructions. Hybridization of MGP, OPN, and ON mRNAs
was performed at 50°C for 16 hours, and the signals were detected by
using a nucleic acid detection kit (Boehringer Mannheim
Biochemica). Controls included hybridization with the sense probes and
RNase treatment prior to hybridization as well as not using the
antisense RNA probe or the anti-digoxigenin antibody. None of the three
experiments showed any positive signals.
Immunohistochemistry
Immunohistochemistry was performed by using serial sections. The
sections were incubated in 0.3% H2O2 in
methanol for 30 minutes followed by a wash in 0.01 mol/L
phosphate-buffered saline and treatment with 1% normal mouse serum for
30 minutes at room temperature to block nonspecific binding by the
antibody. The slides were then incubated with the primary antibodies
for 18 hours at 4°C. The primary antibodies used in this study were
the mouse MAbs 1A423 (DAKO) and MPIII10124
(Developmental Studies Hybridoma Bank, Johns Hopkins University,
Baltimore, Md). 1A4 MAb recognizes 
–smooth muscle actin, which is
present in smooth muscle cells. MPIII101 MAb recognizes rat OPN.
Binding of MAbs was demonstrated by using the Vectastain ABC kit
(Vector Laboratories) according to the manufacturer's instructions.
For negative controls the sections were incubated with either nonimmune
mouse serum instead of the primary antibody or phosphate-buffered
saline instead of the secondary antibody. For preabsorption testing of
MPIII101, the section was incubated with the antibody solution
preabsorbed by OPN protein instead of MPIII101 primary antibody.
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Results |
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We performed Northern blotting of aortic rings from 3-month-old male rats to examine whether changes in the expression of MGP, OPN, and ON mRNAs could be observed during 72 hours of incubation. MGP mRNA was strongly expressed by aortic rings before incubation, and the expression increased gradually during 72 hours of incubation (Fig 1A
). The expression of OPN mRNA was not detected in the
aortic rings before incubation; a weak but apparent expression was
first detected at 5 hours of incubation with increased expression
thereafter (Fig 1B). ON mRNA was strongly expressed by the aortic rings
before incubation, and its strong expression continued for 12 hours of
incubation. Although the expression of ON mRNA decreased thereafter, it
was apparent even after 72 hours of incubation (Fig 1C).
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Next, we carried out Northern blotting of aortic rings from both older
(10-month-old; Fig 2) and younger (2-week-old; Fig 3) rats to examine whether age-specific changes were
observed in the expression of MGP, OPN, and ON mRNAs during 72 hours of
incubation. In the aortic rings from 10-month-old rats, the expression
patterns of MGP and OPN mRNAs were similar to those from 3-month-old
rats (Fig 2A and 2B), although the expression of OPN mRNA at 5 hours of
incubation from 10-month-old rats was slightly weaker than that from
3-month-old rats. In contrast, the expression of ON mRNA in the aortic
rings from 10-month-old rats was apparently weaker than that from
3-month-old rats both before incubation (Fig 4) and at
every incubation period (Fig 2C). After 72 hours of incubation, the
expression of ON mRNA was hardly detectable in the aortic rings from
10-month-old rats. In the aortic rings from 2-week-old rats, the
expression patterns of MGP and OPN mRNAs were similar to those from 3-
and 10-month-old rats (Figs 3A, 3B, and 5), although the
expression of OPN mRNA in the aortic rings from 2-week-old rats was
first detected at 3 hours of incubation. In contrast, the expression of
ON mRNA in the aortic rings from 2-week-old rats was much stronger than
that from 3-month-old-rats both before incubation (Fig 4) and at every
incubation period (Figs 3C and 5).
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To localize the expression of MGP, OPN, and ON mRNAs and to determine
the type of MGP, OPN, and ON mRNA–expressing cells in the aortic
rings, a combination of in situ hybridization and immunohistochemistry
was performed. In the preincubation aortic rings from 3-month-old rats,
MGP and ON mRNAs were readily detected throughout the aortic media (Fig 6A, 6B, 6D, 6U, and 6W), but OPN mRNA was not detected
(Fig 6C). MGP and ON mRNA–expressing cells were identified as VSMCs in
the aortic wall by staining with MAb 1A4 (Fig 6E). At 1 and 3 hours of
incubation the expression of MGP, ON, and OPN mRNAs in aortic rings was
quite similar to that before incubation. At 5 hours of incubation the
expression was similar to that before incubation (Fig 6F, 6G, 6I, and 6J), but a few cells that strongly expressed OPN mRNA appeared (Fig 6H). OPN mRNA–expressing cells were identified as VSMCs by the
staining with 1A4 MAb (Fig 6H and 6J). At 12 hours of incubation the
expression of MGP mRNA was similar to that before incubation (Fig 6K, 6L, and 6O), but the expression of ON mRNA became similarly weak in all
the VSMCs (Fig 6N). On the other hand, the number of VSMCs that
strongly expressed OPN mRNA increased (Fig 6M and 6O). At 24, 48, and
72 hours of incubation the expression of MGP mRNA by VSMCs remained
strong (Fig 6P, 6Q, 6T, 6U, and 6X). The number of OPN mRNA–expressing
VSMCs increased dramatically (Fig 6R, 6T, and 6V), and almost all VSMCs
expressed OPN mRNA strongly. ON mRNA–expressing cells could not be
detected (Fig 6S). In the aortic rings from 10-month- and 2-week-old
rats, the localization of MGP and OPN mRNA–expressing cells was
similar to that from 3-month-old rats at every incubation period. In
the aortic rings from 10-month-old rats, the signals of ON mRNA before
incubation were weaker than those from 3-month-old rats, and they were
hardly detectable even at 5 hours of incubation (data not shown). In
the aortic rings from 2-week-old rats, the signals of ON mRNA before
incubation were stronger than those from 3-month-old rats, and they
were detectable by 24 hours of incubation (data not shown).
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We compared the localization of OPN mRNA with that of OPN protein by
the combination of in situ hybridization and immunohistochemistry. The
results in the aortic rings from 10-month-old rats were similar to
those from 3-month- and 2-week-old rats. OPN mRNA was not detected in
the VSMCs of aortic rings before incubation (Fig 7A).
OPN protein was hardly detectable in most of VSMCs before incubation
(Fig 7B), although very weakly stained VSMCs were observed in some
areas. Both the expression of OPN mRNA and the production of
OPN protein strongly increased at 24 hours of incubation, and the
population of OPN protein–positive VSMCs was quite similar to that of
OPN mRNA–expressing VSMCs (Fig 7C and 7D). The results of negative
controls obtained by using a sense probe for OPN mRNA and preabsorbed
solution for immunohistochemistry of MPIII101, respectively, are also
shown (Fig 7E and 7F).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We described the incubation time–dependent alteration of the expression of MGP, OPN, and ON mRNAs in cultured aortic rings. Northern blotting showed that the expression patterns of MGP, OPN, and ON mRNAs differed. Moreover, we demonstrated that the aortic rings from 3-month-, 10-month-, and 2-week-old rats differed in the expression of ON mRNA but not MGP or OPN mRNAs.
In some cases, such as aortic rings from 3-month-old rats at 24, 48, and 72 hours of incubation, we could not detect ON mRNA by in situ hybridization in spite of the detection of ON mRNA by Northern blotting. There is a possibility that the technique of in situ hybridization or ON probe that we used was inadequate. However, we could detect the signals of rat ON mRNA by using in situ hybridization with the same technique and probe even when Northern blotting showed much weaker signals.19 Therefore, we believe that the technique and probe used were adequate to detect rat ON mRNA signals. We assumed that in situ hybridization was less sensitive than Northern blotting in the case of diffuse expression in many cells with less copy number per cell. Therefore, we consider that the similarly decreased expression of ON mRNA in all VSMCs is a main cause of the failure of ON mRNA detection by in situ hybridization. On the other hand, we could not detect OPN mRNA in aortic rings before incubation in spite of the detection of OPN protein by immunohistochemistry. Since MAb MPIII101 for rat OPN protein is specific, as the preabsorption test indicated, this is probably due to the lesser sensitivity of in situ hybridization compared with immunohistochemistry. In fact, we could detect the signal of OPN mRNA in aortic rings before incubation by Northern blotting with longer exposure (data not shown).
Shanahan et al4 report that late-passage dedifferentiated VSMCs preferentially express MGP and OPN mRNAs in a dispersed culture system. Giachelli et al5 6 report that injury to the adult rat aorta by balloon catheter initiates a time-dependent increase in both OPN protein and OPN mRNA expression in VSMCs. In the present study, both the expression of MGP and OPN mRNAs and the synthesis of OPN protein increased during the 72-hour incubation. Therefore, this in vitro culture system using rat aortic rings seems to reflect both an in vitro dispersed culture system and the in vivo conditions of vascular injury or repair.
Because VSMCs in aortic rings after incubation are considered to be similar to those of in vivo injured or repaired aorta, they appear to function under remodeling conditions. Although strong expression of ON mRNA is reported in remodeling tissues,25 in the present study VSMCs in aortic rings after incubation expressed less ON mRNA than those before incubation. The expression of ON mRNA in VSMCs might respond specifically to the injury. Our previous report7 that the expression of ON mRNA decreases in human aortas when atherosclerosis develops might support this idea. However, it is also possible that the expression of ON mRNA in this incubation system might be specific. The examination of the expression of ON mRNA both in a dispersed culture system and the in vivo conditions of vascular injury or repair appears to be necessary.
Raines et al8 report that ON interacts with platelet-derived growth factor and inhibits the binding of platelet-derived growth factor to its receptor. The expression of ON mRNA decreases incubation time dependently and age specifically in this in vitro culture system. Therefore, the decreased expression of ON mRNA after the injury or in old age might suggest that the repair reaction in the aortic wall to injury occurs easily through the binding of platelet-derived growth factor to VSMCs.
In addition to bone matrix proteins such as MGP,26
OPN,6 7 27 28 and ON,7 8 Bostrom et
al29 report that bone morphogenetic protein-2a is
present within human arterial tissues. These findings
seem to imply that vascular and bone tissues might share common
mechanisms. Although MGP, OPN, and ON are considered to be calcium
regulators in bone and cartilage, we could not find calcium deposition
in the aortic rings of this culture system (H.H. and S.H., unpublished
data, 1994). Among these bone-related factors, calcium-binding acidic
phosphoprotein, OPN, is considered to play an important role for the
mineralization of the tissues. Since OPN might play a role for
mineralization only under certain conditions,30 we
consider that OPN in the rat arterial wall might work for a
function other than mineralization. OPN contains an RGD cell-adhesion
motif that is found in many cell-matrix adhesion molecules, including
fibronectin, thrombospondin, and
vitronectin.12 It also interacts with an
integrin, the vitronectin receptor
(
ß3).31 Therefore, OPN may possibly facilitate
RGD-dependent adhesion and migration of VSMCs.32 The
cell-adhesion properties of MGP appear to be mediated by its
calcium-binding Gla residues. However, its adhesion properties in vitro
were inhibited by synthetic RGD-containing peptides.33
This suggests the possibility of an interaction between OPN and MGP in
mediating cell adhesion. Although the function of OPN and MGP in the
vessel wall is unknown, the increased and simultaneous
expression of their mRNAs after incubation might support the hypothesis
that they cooperatively play roles in the cellular reaction to vascular
injury through their activities as adhesion molecules.
In the present study we incubated aortic rings in serum-free media to avoid the influence of factors in serum. Even after 72-hour incubations we could detect a strong expression of OPN and MGP mRNAs. Our results with tissue viability after 72-hour incubation in serum-free media was consistent with the results of Hosoi et al.18 Moreover, we showed the correspondence of the immunolocalization of OPN protein with the localization of OPN mRNA, suggesting that not only mRNA expression but also rapid protein synthesis were induced in this culture system. The use of metabolically intact aortic rings, which maintain cell-to-cell and cell-to-ECM interactions in a manner that is perhaps more physiologically realistic than that present in cell culture, offers a potentially useful system for characterizing the regulatory mechanisms and functions of these bone matrix proteins in the vessel wall.
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Selected Abbreviations and Acronyms |
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Received December 17, 1994; accepted June 15, 1995.
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