© 1997 American Heart Association, Inc.
Articles |
Identification of Osteoglycin as a Component of the Vascular Matrix
Differential Expression by Vascular Smooth Muscle Cells During Neointima Formation and in Atherosclerotic Plaques
From the University of Cambridge, Department of Medicine, Addenbrooke's Hospital, and the Department of Pathology, Papworth Hospital, Cambridgeshire (N.R.B.C.), UK.
Correspondence to Dr C.M. Shanahan, Department of Medicine, Addenbrooke's Hospital (Box 157), Hills Road, Cambridge, CB2 2QQ UK. E-mail cs131{at}mole.bio.cam.ac.uk
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Abstract |
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Abstract Using differential cDNA screening, we demonstrated that the bone-associated glycoprotein osteoglycin was highly expressed in differentiated adult rat vascular smooth muscle cells (VSMCs) but downregulated in VSMCs that had undergone proliferation in vitro. Further experiments in vitro revealed that osteoglycin gene expression was downregulated by a number of cytokines expressed in vivo (often in association with vascular injury) including basic fibroblast growth factor, transforming growth factor-ß, platelet-derived growth factor, and angiotensin II. In the normal adult rat carotid artery, osteoglycin was expressed in both the media and adventitia. However, osteoglycin mRNA expression was substantially increased in the adventitia and neointima 14 days after balloon injury, implying a role for this protein in vessel remodeling. Northern analysis of mRNA from neonatal rat aortas demonstrated upregulation of osteoglycin mRNA at week 2, after VSMC proliferation had ceased and when matrix modeling was maximal. In situ hybridization studies in human coronary arteries showed that osteoglycin mRNA was expressed by normal medial VSMCs but was downregulated in a subset of intimal VSMCs. Osteoglycin was not expressed in the VSMCs of adventitial vessels but was expressed in a subset of adventitial cells. This expression pattern contrasted with that of SM22
, a contractile
protein marker of VSMC differentiation, which was highly expressed in
the media of all vessels. These data indicate that osteoglycin is a new
marker of differentiated VSMCs and may be an essential component of the
normal vascular matrix.
Key Words: osteoglycin • leucine-rich repeat • matrix • SM22 • atherosclerosis
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Introduction |
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Adult VSMCs in the media of an intact vessel wall exist in a fully differentiated contractile phenotype. In contrast, intimal VSMCs involved in vascular diseases such as atherosclerosis exhibit a less differentiated phenotype, which bears many similarities to that displayed by fetal or neonatal VSMCs.1 2 3 When adult VSMCs are dispersed into cell culture, they spontaneously undergo a phenotypic change such that the phenotype of passaged, cultured VSMCs more closely resembles that of intimal VSMCs in vivo.4 We hypothesized that the genes most strongly expressed by differentiated contractile VSMCs would code for proteins involved in the contractile function of VSMCs and maintenance of the integrity of the healthy vessel wall, while genes expressed by proliferating, dedifferentiated VSMCs might code for proteins involved in the pathogenesis or progression of vascular disease. Using differential cDNA screening, we identified a group of genes whose expression was high in freshly dispersed VSMCs and rapidly downregulated when the cells proliferated in cell culture.5 Consistent with our hypothesis, many of these genes coded for known contractile proteins, eg,
and 
smooth muscle actin, calponin, and SM22, and in situ hybridization
studies confirmed much higher expression of these genes in normal
medial VSMCs in intact human vessels than in disease-associated intimal
VSMCs.5 6 In addition to known genes, our cDNA screen also
identified 4H3 in this group whose identity was unknown. In this study we report the cloning and characterization of 4H3, which we have identified as the rat homologue of osteoglycin, a matrix glycoprotein previously found only in bovine and human bone.7 8 Osteoglycin is a member of a large family of structurally related proteins characterized by tandem repeats of a leucine-rich sequence (LRRs) bounded by two conserved cysteine clusters that are involved in protein-protein interactions.9 10 A subset of these proteins, which includes those most closely related to osteoglycin, is of particular interest because of their ability to bind growth factors, especially members of the TGFß superfamily, via their core proteins.11 Osteoglycin itself was originally named osteoinductive factor because initial isolates of the protein were found to induce ectopic bone formation when inoculated into rat skeletal muscle.12 13 However, this activity was later ascribed to contamination with TGFß-like bone morphogenetic proteins 2 and 3.14 These proteins, which can induce ectopic bone, had copurified with the osteoglycin protein under a number of conditions, suggesting that they were associated in vivo.14
The matrix produced by VSMCs has been shown to be of fundamental
importance in maintaining the contractile phenotype of VSMCs
and in the remodeling process that occurs after vascular
injury.1 15 16 Therefore, we undertook studies to
determine the expression of osteoglycin by VSMCs in vitro, during
development of the rat aorta, after balloon injury to the adult rat
carotid artery, and in the normal and diseased human vessel wall. In
addition, we compared the expression and regulation of osteoglycin in
vivo and in vitro with SM22, a contractile protein marker of VSMC
differentiation. This revealed substantial
heterogeneity of VSMC gene expression in
atherosclerotic vessels, which is most probably associated with
differential regulation of contractile and matrix protein–encoding
genes.
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Methods |
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DNA Sequencing
A cDNA clone approximately 3.2 kb in length and designated 4H3 was isolated from a rat aortic smooth muscle cell cDNA library (
Zap)
by differential screening as previously described.5 4H3
was subcloned into M13p18 and sequenced using the dideoxy
chain–termination method with Sequenase (US Biochemical Corp)
according to the manufacturer's instructions. Full-length sequences
were generated using random sonicated clones and were assembled using
the Staden sequence assembly program.17 18 Protein and
nucleic acid alignments were made using the FASTA
algorithm.19
Culture of Rat VSMCs
Primary cell cultures were established from thoracic and
abdominal aortas excised from 12-week-old (adult) Wistar rats. The
tunica adventitia and endothelium were removed and the
resultant tunica media was dispersed enzymatically as previously
described.20 Dispersed cells were plated at a density of
8x104 cells/cm2 in Dulbecco's modified
Eagle's medium and 10% FCS (Sigma Chemical Company, Ltd). The cells
used in the differential screening experiment have been described
previously.5 Briefly, RNA was harvested either from
confluent cells 7 days after plating (D7 cells) or from cells that had
been subcultured through at least 12 passages. The culture conditions
were such that known markers of the differentiated phenotype
(smooth muscle myosin heavy chain and -smooth muscle actin) were
expressed at high levels in the D7 cells but at lower or undetectable
levels in the passaged cells. RNA was also harvested from confluent
cultures of Rat-2 fibroblasts and used as a control on Northern blots.
For experiments analyzing osteoglycin expression in vitro,
postconfluent aortic smooth muscle cells between passages 6 and 9 were
used. These cells were derived from dispersed cells (as above) passaged
(at a dilution of 1:2) when the cells had reached confluence
(approximately every 4 days) with a medium change every 2 days.
Postconfluent cells were obtained 6 to 7 days after the last passage
(ie, at least 2 days postconfluence) when they were serum deprived for
48 hours. After serum deprivation, cells were restimulated
with 10% FCS or one of the following: 10 ng/mL human bFGF, 10
ng/mL human PDGF, both A and B isoforms (R&D Systems),
10-6 mol/L human synthetic
angiotensin II (Sigma Chemical Company, Ltd), and 10
ng/mL human recombinant TGFß (Austral Biologicals). RNA was
harvested 24 and 48 hours after restimulation.
RNA Preparation and Northern Analysis
Cells were prepared for RNA extraction either by trypsinization
of cultured cells or enzyme dispersion of aortic medial VSMCs from
neonatal or adult rats. Total cytoplasmic RNA was isolated from these
cells and a variety of rat tissues by NP-40 lysis as previously
described.5 RNA (10 to 30 µg) was electrophoresed in
1.5% agarose gels containing 2.2 mmol/L formaldehyde in a
buffer containing 20 mmol/L MOPS, 1 mmol/L
EDTA, 5 mmol/L sodium acetate, and 0.5µg/mL
ethidium bromide. The integrity of the RNA was visualized by UV
illumination of gels before and after transfer to Hybond-N as specified
by the manufacturer. Probes were generated and filters hybridized,
washed, and exposed as described previously.5 Filters were
reprobed with GAPDH to check RNA integrity and loading.
Balloon-Injury Model
Adult male Wistar rats (>300 g) were anesthetized with
ketamine and xylazine and an injury to the intima of the left
common carotid artery was produced with an inflated balloon catheter (2
French, Baxter Health Care) as previously described.21
Seven or 14 days, later both the left and right carotid arteries were
dissected and processed as described above for human coronary
arteries.
Human Coronary Arteries
Fresh human coronary arteries (n=11) were obtained from
four recipient hearts excised at the time of orthotopic
transplantation. Samples were mounted in Tissue-Tek OCT embedding
compound (Miles Ames Division, Inc), snap-frozen in liquid nitrogen,
and subsequently stored at -70°C before sectioning. Sections were
cut (8 to 10 µm) and mounted onto gelatinized slides, refrozen,
and stored at -70°C until study by in situ hybridization and
immunohistochemistry.
Immunohistochemistry
Immunohistochemistry was performed on serial sections using a
horseradish peroxidase-based detection system (ABComplex/HRP, Dako)
according to the manufacturer's instructions. VSMCs were identified
with a mouse monoclonal antibody to human -smooth muscle actin
(Dako, M815, dilution 1:25). Macrophages were identified with a
mouse monoclonal antibody to CD68 (Dako, macrophage: EMB11,
dilution 1:20), which stains all tissue macrophages. Calcium
was identified in lesions by using von Kossa stain.
In Situ Hybridization
In situ hybridization to sections of rat carotid artery was
carried out as previously described, using 35S-UTP-labeled
sense and antisense cRNA probes generated by in vitro transcription
from the T7 and T3 promoters of Bluescript SK-linearized plasmid
containing the 3.2-kb 4H3 cDNA.6 In situ hybridization to
human tissue was carried out on serial sections adjacent to those used
for immunohistochemistry according to the method of Wisden et al
22 using a 35S-labeled
oligonucleotide corresponding to the first 45
nucleotides of the published human osteoglycin cDNA
sequence.7 22 Negative controls included predigestion of
the sample with RNAse and inclusion of 20-fold excess of unlabeled
oligonucleotide to compete with labeled probe. After
washing, sections were coated in Ilford K5 emulsion and left to expose
at 4°C in the dark for between 3 and 6 weeks. Sections were developed
in Kodak D19 developer, fixed, and then stained with hematoxylin/eosin.
In situ hybridization was carried out on the same vessels to determine
expression of SM22 (a positive marker of differentiated SMCs) using
35S-UTP-labeled sense and antisense cRNA probes as
previously described.6
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Results |
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Sequence and Northern Analysis of cDNA Clones
Northern analysis of cDNA clones isolated from the differential screening of the D7 library identified one clone, designated 4H3, that was highly expressed in freshly dispersed aortic cells but only weakly expressed in cultured rat aortic VSMCs and Rat-2 fibroblasts (Fig 1A
). On Northern blots,
4H3 hybridized to a single major band approximately 3.5 kb in size
present in aortic RNA. 4H3 was highly expressed in aorta, uterus,
and kidney and was expressed at very low levels in lung and ovary (Fig 1B).
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Sequencing of 4H3, a 3.2-kb cDNA clone, and comparison of the sequence
with the EMBL/GENBANK databases revealed that this clone had
significant homology (approximately 86%) to human and bovine
osteoinductive factor (renamed osteoglycin).7 A single
open reading frame of 897 bp could be identified, and from this nucleic
acid sequence, an amino acid sequence was deduced encoding a putative
protein of 298 amino acids with a predicted molecular weight 
34 000
D (Fig 2A). This amino acid sequence is
86% homologous over the entire 298 amino acids with the human
osteoglycin precursor and 85% homologous with the bovine osteoglycin
precursor protein. This level of homology suggests that 4H3 encodes the
rat homologue of these proteins. The rat protein contains a single
N-glycosylation site at
258N-X-T260.
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Rat osteoglycin also shares homology with a number of proteins that
contain LRRs flanked by two cysteine clusters. It is most closely
related to chicken proteoglycan-Lb, with 48% homology in the
C-terminal portion of the protein (aa 86 to 296) including
100% conservation of the position of the two cysteine clusters
bounding the LRR sequences.23 Osteoglycin also shares
sequence homology with decorin and biglycan (30%). However, these
proteins do not share 100% conservation of the cysteine
residues.24 Analysis of the protein sequence of
rat osteoglycin revealed that it contained six LRRs. Fig 2B
shows the
consensus sequence for rat osteoglycin LRRs and compares it with the
general consensus from other related proteins.
Regulation of Osteoglycin Expression in Rat Aortic SMCs In
Vitro
The reduced expression of osteoglycin in cultured rat aortic SMCs
compared with freshly dissociated aortic SMCs suggested an
inhibitory effect of cell-cycle entry or
mitogenic stimulation, or both, on osteoglycin expression.
We therefore wished to determine the effects of mitogenic
stimulation on osteoglycin expression. For practical reasons, such
experiments could not be performed on freshly dispersed VSMCs, in which
the effects of enzyme dispersion cannot be dissociated from the effects
of mitogenic stimulation. Therefore, for these experiments,
we used postconfluent, stationary phase passaged VSMCs that still
expressed sufficient osteoglycin mRNA to be detectable by Northern
analysis but no longer proliferated, even in the presence of
growth factors. Postconfluent rat VSMCs were serum starved for 48 hours
before restimulation with FCS or single mitogens as described in
"Methods." RNA was harvested before and during serum starvation,
and 24 and 48 hours after addition of growth factors and osteoglycin
expression was detected by Northern analysis. Serum-deprived
VSMCs expressed higher levels of osteoglycin mRNA throughout the period
of deprivation than serum-stimulated confluent cells at
time 0 (ie, before serum withdrawal). Treatment with either single
factors (TGFß1, bFGF, PDGF, or angiotensin II) or FCS
resulted in downregulation of osteoglycin mRNA 24 and 48 hours later
compared with the levels in serum-deprived cells. This pattern of
expression was similar to that of elastin, another vascular matrix
protein, but contrasted with that of SM22, which was not
significantly altered by the addition of cytokines under the
same experimental conditions (Fig 3).
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Osteoglycin Expression After Rat Carotid Injury
The expression of osteoglycin was analyzed by using in
situ hybridization to rat carotid arteries before and after
balloon-induced intimal injury. In uninjured carotid arteries,
osteoglycin mRNA was expressed at a low level throughout the media but
was more highly expressed in adventitial cells (Fig 4). Seven days after injury, there was
detectable osteoglycin mRNA expression in neointimal VSMCs,
which increased substantially at 14 days, particularly in cells closest
to the luminal edge. By comparison, medial expression remained modest
throughout. At both 7 and 14 days after injury, the adventitia had
become thicker compared with the uninjured control vessel, and there
was substantial upregulation of osteoglycin mRNA expression in a subset
of adventitial cells.
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Osteoglycin Expression During Rat Aortic Development
Northern blot analysis was used to study expression of
osteoglycin in the postnatal aorta. This showed barely detectable
levels of osteoglycin mRNA at day 2 with a gradual increase in
osteoglycin expression throughout postnatal development, demonstrated
by incremental increases through weeks 2 and 4 to maximal levels in the
adult at week 12 (Fig 5).
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Osteoglycin Expression in Human Coronary Arteries
To determine the distribution of osteoglycin mRNA in normal and
diseased human vessels, in situ hybridization was performed on frozen
sections from 11 coronary arteries. These sections included
areas of normal vessel with intimal thickenings composed entirely of
VSMCs, fatty streaks characterized by intimal lipid accumulation and
focal, eccentric intimal thickening with macrophage
infiltration, and complex atherosclerotic plaques characterized by a
necrotic lipid-filled core with associated macrophages and
calcification covered by a VSMC-rich fibrous cap. In a number of these
vessels, the media adjacent to the atheromatous plaque
was thinned. The Table summarizes the
composition of the vascular lesions studied and compares the expression
pattern for osteoglycin and SM22 in each section. Osteoglycin mRNA
was highly expressed in the media and intima of normal coronary
arteries, although expression in intimal VSMCs of diffuse intimal
thickenings was often patchy and confined to a subset of VSMCs. This
expression pattern contrasted with that of SM22, which was
downregulated in intimal VSMCs (Fig 6).
Osteoglycin was also expressed by medial VSMCs in small vessels with an
identifiable elastic lamina. However, it did not appear to be highly
expressed in VSMCs of adventitial vessels (vasa vasora). These small
vessels were positive for -SM actin and also expressed SM22 mRNA
(Fig 7). In advanced lesions, expression
of osteoglycin was mainly confined to medial VSMCs, with some low,
patchy expression in the intima, even in areas containing large numbers
of VSMCs (Fig 8). Any intimal expression
detectable was usually in cells close to the internal elastic lamina
and the occasional VSMC in the fibrous cap (not shown). In sections of
advanced atherosclerotic lesions, osteoglycin was expressed at a lower
level in the media than in normal vessels. This finding contrasted with
SM22 expression, which remained high in the media of vessels with
advanced lesions but was downregulated in most intimal cells. In the
adventitia, osteoglycin was expressed by a small subset of -SM
actin–negative cells. The subset of adventitial cells that expressed
osteoglycin exhibited no particular localization pattern in relation to
the vessel (Fig 9). Osteoglycin mRNA
expression was not detectable in macrophages or
endothelial cells (not shown), nor was the expression
of osteoglycin mRNA associated with calcification within
atherosclerotic plaques.
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Discussion |
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We previously identified the cDNA 4H3 because of its higher expression in primary cultured adult rat VSMCs than in equivalent cells that had undergone 12 passages in culture.5 Subsequent Northern analysis demonstrated even higher expression of 4H3 in freshly dispersed adult aortic VSMCs that had not been subjected to cell culture, indicating that 4H3 represented a marker for the mature, differentiated phenotype of VSMCs. In this study we have shown that 4H3 encodes rat osteoglycin and contains six tandem repeats of a 24–amino acid conserved leucine-rich domain. The protein also contains some sequences with similarities to the glycosaminoglycan attachment site; however, previous purification studies of the bovine protein suggest that all attached polysaccharides are N glycosylated.13 24 Our studies confirm that osteoglycin is not a bone-specific protein, as its name suggests, since it is also highly expressed in the vasculature, uterus, and kidney.
Although 4H3 was originally identified using a cell-culture model, it
is clear that expression of osteoglycin is markedly reduced in cultured
VSMCs. This loss of expression in cell culture may have been caused by
the individual or combined effects of withdrawing the cells from their
extracellular environment, of mitogenic stimulation, or of
entry of the cells into the cell cycle. By studying cells that had
grown to confluence before serum deprivation, we were able
to define the effects of mitogens on gene expression independently of
proliferation, since restimulation of postconfluent VSMCs with single
growth factors does not result in proliferation of the
contact-inhibited cells. We found that serum withdrawal caused an
upregulation of osteoglycin expression that was suppressed by the
growth factors studied. We obtained similar results when we studied
expression of another VSMC matrix protein, elastin, while the same
growth factors had no effect on gene expression of the contractile
protein SM22. These data confirm that growth factors can inhibit
expression of osteoglycin in vitro where expression is already
relatively low. Furthermore, since a number of the growth factors and
cytokines tested are released at the site of vascular injury by
platelets, VSMCs, and macrophages, these factors may exert
a similar effect on osteoglycin expression in both rat and human
vessels in vivo.25 26 27 Indeed, this factor may account for
the localized downregulation of osteoglycin expression throughout
atherosclerotic vessels.
The injury produced by balloon catheterization and the subsequent response in the rat carotid artery has been well characterized.21 The balloon causes endothelial denudation and medial VSMC death. This precipitates medial VSMC proliferation followed by migration of medial VSMCs into the intima where they proliferate. Intimal VSMC proliferation is maximal approximately 7 days after injury. Thereafter, intimal VSMC proliferation rapidly reduces and the neointima progresses by matrix deposition and remodeling.21 We used in situ hybridization to study osteoglycin mRNA expression in this model, since, in contrast with Northern analysis, it provides details on the topographical distribution of gene expression. This procedure demonstrated that osteoglycin expression was modestly upregulated in intimal VSMCs 7 days after injury and substantially upregulated in intimal VSMCs 14 days after injury. Thus, the greatest increase in osteoglycin gene expression occurred after most intimal VSMC proliferation had ceased. The high intimal expression of osteoglycin 14 days after balloon injury is similar to that observed for tropoelastin, whose expression is increased in cells that have ceased proliferating in vivo.28 Taken together, these data are consistent with a phase of matrix gene expression that follows VSMC proliferation in the response to vascular injury.
The high adventitial expression of osteoglycin was unexpected, since
our initial Northern analysis had indicated that osteoglycin
was not highly expressed by fibroblasts in culture. However, recently
it has become clear that the adventitia contributes substantially to
the response to vascular injury. In particular Shi et al29
have shown that after severe balloon-induced injury, some adventitial
cells express several VSMC-specific genes including -SM actin and
desmin. These observations have lead to the suggestion that adventitial
fibromyoblasts can "differentiate" into VSMCs, which may contribute
to neointima formation by migrating through breeches in the
media caused by the balloon.29 Our findings highlight the
effects of balloon injury on induction of adventitial matrix
deposition, which may make an important contribution to vessel
remodeling, and are consistent with the notion that adventitial
myofibroblasts are involved in vessel repair. However, further studies
are required to determine whether osteoglycin-expressing adventitial
cells contribute to formation of the neointima.
The in situ hybridization studies also showed that there was much higher osteoglycin expression in the intima and adventitia than in the media of injured vessels. However, due to limitations in quantifying expression levels using in situ hybridization, it was not possible to determine whether the low relative level of medial expression represented a real downregulation of gene expression in the injured media compared with medial expression in the uninjured vessel.
A number of lines of evidence have suggested that VSMCs involved in the repair of the adult vascular wall reexpress an immature phenotype characteristic of proliferating fetal or neonatal VSMCs.2 3 However, our data so far had indicated that neointimal VSMCs expressed high levels of osteoglycin, a gene that we had already shown was most highly expressed by mature adult VSMCs, and that osteoglycin expression appeared to increase after cell proliferation has ceased. We were therefore interested to study the developmental regulation of osteoglycin. During postnatal aortic development, most VSMC proliferation ceases by week 2, and the vessel matures thereafter by a combination of VSMC hypertrophy and deposition of extracellular matrix, particularly elastin and collagen.3 28 30 31 Postnatal vascular development therefore comprises an early proliferative phase followed by a longer maturation phase. We found that osteoglycin expression was low during the first 2 weeks of development and increased toward adult levels after week 4. This pattern of osteoglycin expression suggests that osteoglycin is not required for the proliferative phase of vascular development and indicates an important role for osteoglycin in the development and maintenance of the mature vascular matrix. Again, there are some similarities between expression of osteoglycin and elastin, whose expression also increases over the same period of postnatal aortic development before the formation of complete elastic lamina in the mature vessel.3 28 31 Expression of both genes by intimal VSMCs after the proliferative response to vascular injury implies an attempt by such cells to repair the vessel wall by the deposition of appropriate matrix components.
The above in vitro and in vivo data on rat VSMCs indicated that
osteoglycin expression was required for the maintenance of the
normal mature vascular wall. Consistent with these data, we
found that osteoglycin was expressed by medial VSMCs and some intimal
VSMCs in normal human vessels but was downregulated in most intimal
VSMCs associated with atherosclerotic lesions. It has long been
recognized that intimal VSMCs in atherosclerotic plaques have reduced
expression of genes for contractile proteins and contain fewer
contractile filaments than their medial counterparts. Such
"dedifferentiated" cells were thought to contribute to the
development of the atherosclerotic plaque. Our data indicate that
intimal cells may also lack the capacity to synthesize matrix
components that could contribute to plaque stability. The differential
expression of osteoglycin and SM22 in the intimal and medial VSMCs
highlights the heterogeneity of VSMC phenotype
in lesions and suggests that although osteoglycin and SM22 are both
VSMC differentiation markers, they are not coordinately regulated. The
lack of expression of osteoglycin mRNA in coronary adventitial
vessels, which clearly contained VSMCs, may indicate a lower matrix
turnover in these vessels or qualitative differences in the matrix
produced by elastic and nonelastic arteries. There may be unique
properties of vasa vasora with respect to other small arteries, and
this possibility deserves further analysis. There was no
association between osteoglycin expression and calcification in
lesions. Some association may have been expected, as osteoglycin was
originally isolated from bone and other bone-associated proteins such
as osteopontin and matrix Gla protein, which have been implicated in
vascular calcification.6
Our data suggest that osteoglycin may be important in developing and maintaining the mature vascular matrix; however, its precise role remains to be determined. Close homologues of osteoglycin, such as chicken proteoglycan Lb, which is expressed in a subset of chondrocytes during cartilage development, and decorin, which is expressed in the developing avian aorta, are thought to contribute to ordering of the matrix by interacting with collagens.23 32 Its homology to known TGFß-binding proteins may also be of relevance, since TGFß has been shown to have significant effects on VSMC behavior, during both normal development and vascular disease.1 27 Further studies are now required to determine the proteins with which osteoglycin interacts in the vessel wall and how these interactions affect development and disease progression.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants to P.L. Weissberg and to C.M. Shanahan from the British Heart Foundation. Dr Shanahan is a British Heart Foundation basic scientist lecturer and Prof Weissberg is a British Heart Foundation professor. We wish to thank Jo Horsley and Carolyn Belcher for assistance with immunohistochemistry, Birgitte Bruun for assistance with tissue culture, and Suzanne Diston for help in preparing the manuscript.
Received February 14, 1997; accepted April 15, 1997.
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