Integrative Physiology |
From the Departments of Pathology (J.W.F., M.G.K., S.L., T.N.W.) and Vascular Surgery (J.W.F., M.M.C., A.W.C.), University of Washington, Seattle.
Correspondence to Thomas N. Wight, PhD, Department of Pathology, HSB-Room 507, Box 357470, 1959 NE Pacific St, Seattle, WA 98195-7470. E-mail tnw{at}u.washington.edu
| Abstract |
|---|
|
|
|---|
Key Words: proteoglycans smooth muscle cells extracellular matrix gene therapy hyperplasia
| Introduction |
|---|
|
|
|---|
80% of the volume of
the intimal lesion after 4 weeks. Moreover, the phenotype and
behavior of SMCs are influenced through interactions between ECM
receptors at the surface of SMCs and specific ECM
ligands.3 4 5 6 Decorin is a member of the family of small leucine-rich proteoglycans.7 8 9 The core protein of decorin contains 1 dermatan sulfate side chain near the amino terminal of the core protein and 2 independent binding domains for collagen type 1 and transforming growth factor (TGF)-ß1.10 Decorin was chosen for the present study because it has previously been shown to inhibit TGF-ß1induced ECM accumulation in a glomerulonephritis model in rats11 and a lung fibrosis model in rabbits,12 to inhibit proliferation of various cell types,13 14 15 16 and to affect collagen fiber formation.17 18 In the present study, we used cell-mediated transfer of the decorin gene to achieve local overexpression of bovine decorin in the neointima that develops after balloon injury of the rat carotid artery. We demonstrate that the local overexpression of this ECM molecule reduces intimal thickening in response to arterial injury by altering the composition of the ECM and reducing ECM volume.
| Materials and Methods |
|---|
|
|
|---|
Cell Seeding
Balloon injury and cell seeding in Fischer 344 rats were
performed as described previously.20 All surgical
procedures were performed according to the Principles of Laboratory
Animal Care and the Guild for the Care and Use of Laboratory
Animals (National Institutes of Health publication No. 86-23,
revised 1985).
Tissue Preparation and Morphometric Analysis
The rats received 50 mg bromodeoxyuridine (BrdU) subcutaneously
24 hours before they were euthanized. Subsequently, the animals were
fixed by perfusion with 10% neutral-buffered formalin at 120
mm Hg pressure, and the BrdU-positive cells were detected with a
monoclonal antibody to BrdU (Boehringer-Mannheim).
Alternatively, tissue was processed for transmission electron
microscopy as described.22 The morphometric measurements
were performed as described previously.22 Cell density was
determined by counting nuclei per high-power field. Histochemical
staining for collagen was performed by using Massons trichrome
procedure.23
Reverse TranscriptionPolymerase Chain Reaction
RNA from injured and uninjured carotid arteries was isolated as
described earlier24 and reverse-transcribed into cDNA by
use of Superscript TN II (Life Technologies). A 180-base DNA fragment
of bovine decorin cDNA was amplified (forward primer 312, 5' AGT GCC
AAA AGA CCT TCC 3', 329; reverse primer 491, 5' AGT CGT TCC AAT TTC ACC
3', 474).
Immunocytochemistry
Polyclonal rabbit antisera to the core proteins of mouse decorin
(LF-113) and bovine decorin (LF-94) and to the human
1 (I) c-telopeptide of collagen I (LF-67) were
generous gifts of Dr Larry Fisher (National Institute of Dental
Research, Bethesda, Md). The rabbit antiserum to human versican was
generously provided by Dr Richard Le Baron (University of Texas at San
Antonio, San Antonio, Tex). The goat polyclonal antisera to human
fibronectin was obtained from ICN Pharmaceuticals Inc. Sections to be
stained for the proteoglycan core proteins of decorin and versican were
digested with chondroitin ABC lyase (ICN Biomedicals) at 200 mU/mL in
0.1 mol/L Tris-acetate, pH 7.3, for 1 hour at 37°C.
Cell Culture
Aortic SMCs from male Fischer 344 rats were obtained as
described previously.20 Transduced cells were used for
experiments between 4 and 8 passages after the initial transduction.
After selection by means of the neomycin analogue G418 (800 µg/mL),
SMCs were maintained in 10% calf serum.
cDNA Probes
The rat versican cDNA probe against the V3 form of SMC versican
was obtained from Joan M. Lemire (University of Washington, Seattle).
Probes were 32P-labeled by random priming
(Amersham Pharmacia Biotech) by use of
5'-(
-32P)dCTP.25
Statistical Analysis
A Student t test combined with a Welch
correction for separate variances was used to determine statistical
differences between LXSN- and LDSN-seeded carotid arteries. A value of
P<0.05 was considered significant.
| Results |
|---|
|
|
|---|
|
Ectopic Expression of Decorin Proteoglycan in Rat SMC In
Vitro
Pooled Fischer 344 rat SMCs transduced with the LDSN retrovirus
were examined for bovine decorin mRNA and core protein expression.
Figure 2B
demonstrates the expression of
bovine decorin mRNA as determined by use of a species-specific DNA
probe. Expression of bovine decorin core protein was demonstrated by
Western blotting (Figure 2C
) with a species-specific antibody to
bovine decorin (LF-94).
|
Long-Term Expression of Bovine Decorin in Injured Carotid Arteries
After Seeding of LDSN-SMCs
Fischer 344 rat SMCs transduced with the LXSN or LDSN construct
were seeded onto the luminal surface of carotid arteries of Fischer 344
rats immediately after balloon injury. The seeded cells adhere to the
luminal surface of the denuded vessel and begin to form an
intima.20 26 Intimas resulting from seeding of LXSN cells
demonstrated immunostaining only for rat decorin in the
adventitia and minimal accumulation in the intima at 4 weeks after
injury (Figure 3a
). As expected, no
staining was observed after the same section was
immunostained with the antibody to bovine decorin, LF-94
(Figure 3b
). Similar results were obtained at 1 and 2 weeks
after seeding of LXSN cells (not shown).
|
The antibody LF-94 to bovine decorin enabled us to discriminate between
endogenous rat decorin and the newly introduced bovine
decorin. Figure 4A
shows a time course of
bovine decorin expression in LDSN-seeded vessels. Strong staining for
bovine decorin was found in the intima at 1, 2, and 4 weeks after
injury, indicating that the cell-mediated transfer of the bovine
decorin gene was successful and led to the expression and accumulation
of bovine decorin in the intima. At 1 and 2 weeks, accumulation of
decorin was also seen in the adventitia. This adventitial accumulation
is inherent to the cell-seeding procedure, because at the end of the
cell seeding, the excess cells that do not adhere to the internal
elastic lamina are flushed into the wound around the vessel. These
cells populate the scar tissue and the adventitia. In addition to
immunocytochemistry, reverse transcriptionpolymerase chain reaction
(PCR) was used to demonstrate mRNA expression of bovine decorin in
LDSN-seeded carotid arteries. For this purpose, mRNA of uninjured,
LXSN-seeded, and LDSN-seeded vessels was isolated 4 weeks after SMC
seeding, reverse-transcribed, and subjected to PCR by using
bovine-specific primers for a 180-bp sequence of decorin cDNA. The
plasmid containing the cDNA for bovine decorin served as positive
control. As shown in Figure 4C
, no PCR product was obtained
from uninjured or LXSN-seeded vessels, whereas a band
representing the 180-bp sequence was obtained from the
LDSN-seeded vessels. These results, together with the
immunostaining for decorin, demonstrate that bovine
decorin mRNA and protein are expressed in the LDSN-seeded vessels up to
4 weeks after injury. In addition, we analyzed the expression
of rat decorin (LF-113) in the same sections (Figure 4B
). Rat
decorin is confined to the adventitia and does not change in the LDSN
cellseeded carotid arteries.
|
In addition, it was determined whether the expression of the bovine decorin in Fischer 344 rats gives rise to antibodies against bovine decorin, which could, in turn, neutralize decorin-mediated effects. For this purpose, SMCs overexpressing bovine decorin were grown in culture, and conditioned medium was used as a source from which bovine decorin was isolated by anion exchange chromatography and ethanol precipitation. Subsequently, these extracts were run on a SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. This membrane was probed with serum obtained from animals 4 weeks after seeding of LDSN cells and LXSN SMCs. A biotinylated goat anti-rat antibody was used for detection. No signal for bovine decorin was obtained with the serum of rats seeded with LDSN cells, suggesting that no antibodies to bovine decorin were produced for up to 4 weeks after seeding (data not shown).
Reduced Intimal Area and Increased Intimal Cellularity in Injured
Carotid Arteries Overexpressing Bovine Decorin
Intimal areas of carotid arteries seeded with LDSN SMCs, compared
with LXSN-seeded arteries, were significantly reduced by 35% at 4
weeks (P<0.01, Figure 5A
). No
significant difference was observed at 2 weeks. To investigate whether
a reduction in intimal SMC proliferation might be responsible for the
reduced intimal areas by decorin overexpression, the BrdU labeling
index was determined at 1 and 2 weeks. At 1 week, the percentage of
BrdU-positive cells was 14.4±0.8% in LXSN cellseeded intimas versus
12.8±1% in LDSN cellseeded intimas (mean±SEM, n=5). At 2 weeks,
the percentage of BrdU-positive cells was 3.0±0.78% in LXSN
cellseeded intimas versus 4.3±0.9% in LDSN cellseeded intimas
(mean±SEM, n=5). These data suggest that intimal SMC proliferation was
not affected by overexpression of decorin.
|
The intimal nuclear density was determined by counting SMC nuclei per
high-power field. As shown in Figure 5B
, intimal nuclear density
was significantly (P<0.01) higher in LDSN cellseeded
vessels at 4 weeks compared with LXSN cellseeded intimas, suggesting
that ECM accumulation might be reduced in decorin-overexpressing
intimas. The observations that the total numbers of nuclei (Figure 5C
) were the same in the intimas of LXSN cell and LDSN
cellseeded vessels but that the intimal area was reduced in LDSN
cellseeded vessels at 4 weeks also indicate that intimal matrix
accumulation was impaired by decorin overexpression.
In addition, at 4 weeks after injury, the luminal cross-sectional area was significantly increased in the carotid arteries overexpressing decorin (LDSN, 0.18±0.06 mm2; LXSN, 0.11±0.03 mm2; n=9, mean±SD).
Decorin Overexpression Induces Alterations in Composition and
Organization of Neointimal ECM
The accumulation of fibronectin and versican in intimas of LXSN
cell and LDSN cellseeded carotid arteries was analyzed by
immunostaining. Four weeks after cell seeding, the
staining for versican and fibronectin was much less in the LDSN
cellseeded vessels (Figures 6g
and 6h
)
than in the LXSN cellseeded vessels (Figures 6c
and 6d
). On
the other hand, the LDSN cellseeded intimas showed more pronounced
staining for collagen, as shown by immunohistochemistry for collagen
type I (Figure 6f
) and by histochemistry using Massons
trichrome stain (Figure 7b
). In most
cases, the increased staining for collagen was pronounced at the base
of the neointima. Transmission electron microscopy showed
these regions to be enriched in collagen fibrils in the
decorin-overexpressing neointimas (Figures 8A
and 8B
), whereas fewer collagen
fibrils were present in the LXSNseeded vessels (Figures 8C
and 8D
). The ECM in these areas of the LXSN-seeded vessels contained
large spaces filled with amorphous electron-dense materials and
occasional bundles of collagen fibrils cut in cross section, as well as
isolated collagen fibrils cut in longitudinal section (Figures 8C
and 8D
). In contrast, virtually the entire ECM at the base of
the neointima in the LDSN-seeded vessels was filled with
collagen fibrils cut in cross section, with occasional immature elastic
fibers associated with the surface of the neointimal SMCs
(Figures 8A
and 8B
). It is of interest that the bulk of the
seeded cells was found at the base of the neointima by 1
month after seeding.20 22 26
|
|
|
| Discussion |
|---|
|
|
|---|
Expression of bovine decorin in the neointima of balloon-injured Fischer 344 rats was accomplished by cell-mediated gene transfer using autologous aortic SMCs that were retrovirally transduced with bovine decorin in vitro. By this method, expression of bovine decorin within the neointima was achieved over the entire experimental period of 4 weeks. The use of the bovine decorin gene provides a clear distinction between endogenous rat decorin and the experimentally introduced bovine decorin by use of the species-specific anti-decorin antibodies LF-113 (rat) and LF-94 (bovine) as well as by use of specific cDNA probes and PCR primers.
Decorin expression caused a significant reduction of intimal area by 35±4% after 4 weeks. The proliferative response after injury was not affected because the percentage of BrdU-labeled cells and the total cell number per intimal cross section were the same in vector control cellseeded and decorin cellseeded intimas. The finding that intimal areas were reduced without a change in total intimal cell number suggests that reduced accumulation of ECM is the mechanism responsible for the inhibition of intimal thickening by decorin. In support of this conclusion, morphometric analysis revealed that the cell density was markedly higher in the decorin cellseeded intimas after 4 weeks. Although the mechanism by which decorin causes these changes is unknown, a possible explanation could be the inhibition of TGF-ß1 activity, in view of the fact that decorin has been reported to inhibit TGF-ß1 activity in various models in vitro30 31 and in vivo.11 12 32 TGF-ß1 is known to be an important component in the regulation of matrix accumulation in response to wound repair33 34 35 36 and is thought to play a similar role in the response to arterial injury.37 38 Decorin does not interfere with the process of TGF-ß1 activation. Instead, the core protein of decorin binds and neutralizes the active TGF-ß1 molecule.31 39 Indeed, although we did not determine TGF-ß1 activity in vivo, the induction of versican mRNA by TGF-ß1 is markedly inhibited in cultured Fischer 344 rat SMCs that overexpress bovine decorin in vitro (authors unpublished data, 1999). This observation suggests that retrovirally expressed decorin can inhibit TGF-ß1 activity. The finding of reduced intimal versican and fibronectin accumulation in the injured carotid arteries seeded with decorin-overexpressing cells supports the hypothesis that TGF-ß1 activity is reduced by decorin because both versican and fibronectin are induced by TGF-ß1 and have, among other ECM molecules, been used to document reduced TGF-ß1 activity in experimental models of kidney and lung fibrosis.11 12 32 Furthermore, antibodies to TGF-ß1 have a similar effect and reduce neointimal hyperplasia by decreasing versican and fibronectin accumulation without affecting cell proliferation after arterial injury in the rat.38 However, our finding that the neointimas seeded with decorin-expressing cells stain intensely for collagen would argue for an additional TGF-ß1independent mechanism, because TGF-ß1 is known to increase collagen synthesis by arterial SMCs.40
The reasons for the reduction of ECM volume and the enrichment of collagen fibrils in decorin-overexpressing intimas are not known. Loss of proteoglycans, such as versican, and other ECM proteins, such as fibronectin, may provide space and concentrate other ECM components, such as fibrillar collagen. It is not known whether total intimal collagen content is changed or whether decorin causes a shift in collagen organization. For example, decorin interacts with collagen types I and II.41 Initially, it was shown that decorin inhibits collagen fibrillogenesis.42 However, other reports now suggest that decorin can also affect collagen fiber formation and arrangement in various ways. Decorin was recently shown to improve the tensile properties of collagen fibers43 and to increase fibril diameter17 in vitro. In vivo, decorin is frequently found to be closely associated with collagen and is thought to control collagen fiber formation and arrangement, in particular, the interfibrillar spacing (for review see Reference 44 ). The distance and the thickness of collagen fibers correlate with the amount of closely associated decorin and biglycan in tendons.45 46 It also may be that the binding of decorin to collagen inhibits collagen degradation and stabilizes the collagen fibrillar network. For example, decorin is resistant to proteolysis during interleukin-1stimulated cartilage catabolism, and this resistance has been correlated with the accumulation of type II collagen.47 Whether decorin influences collagen degradation in blood vessels in response to injury awaits further study. In healthy vascular tissue, decorin is heavily expressed in the adventitia together with type I collagen, whereas its expression in the media is low. Decorin is also found in the vascular lesions in areas enriched in collagen,48 49 50 51 suggesting that decorin is involved in the formation of dense collagen-rich structures naturally as lesions form.
The change in the nature of the ECM in the injured vessels in response to the overexpression of decorin differs significantly from the change in ECM after vascular injury in animals treated with another glycosaminoglycan, heparin.52 Infusion of heparin into animals after balloon injury to the carotid artery causes a significant reduction of intimal collagen and elastin and an increase in proteoglycans. Thus, although both molecules decreased intimal thickening in response to vascular injury, they had dramatically different effects on remodeling the ECM. Such differences are likely to lead to different mechanical properties of the formed lesions.
In conclusion, the local overexpression of the proteoglycan decorin in injured carotid arteries may represent a novel gene therapeutic approach to the reduction of intimal thickening by decreasing ECM volume. The reduction of ECM volume involves the loss of molecules that typify the loose provisional ECM, such as versican and fibronectin, and the enrichment of molecules that contribute to a denser ECM, such as fibrillar collagen. Such conversions are typical of ECM changes seen in during wound healing.53 Whether such changes lead to increased tensile strength of forming lesions and eventually to atherosclerotic plaque stabilization54 55 will require further investigation.
| Acknowledgments |
|---|
| Footnotes |
|---|
Received July 12, 1999; accepted December 30, 1999.
| References |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
K. Fu, M. J. Corbley, L. Sun, J. E. Friedman, F. Shan, J. L. Papadatos, D. Costa, F. Lutterodt, H. Sweigard, S. Bowes, et al. SM16, an Orally Active TGF-{beta} Type I Receptor Inhibitor Prevents Myofibroblast Induction and Vascular Fibrosis in the Rat Carotid Injury Model Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 665 - 671. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. W. Fischer Tenascin-C: A key molecule in graft stenosis Cardiovasc Res, June 1, 2007; 74(3): 335 - 336. [Full Text] [PDF] |
||||
![]() |
R. Huang, M. J. Merrilees, K. Braun, B. Beaumont, J. Lemire, A. W. Clowes, A. Hinek, and T. N. Wight Inhibition of Versican Synthesis by Antisense Alters Smooth Muscle Cell Phenotype and Induces Elastic Fiber Formation In Vitro and in Neointima After Vessel Injury Circ. Res., February 17, 2006; 98(3): 370 - 377. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. A. Spencer, S. L. Hacker, E. C. Davis, R. P. Mecham, R. H. Knutsen, D. Y. Li, R. D. Gerard, J. A. Richardson, E. N. Olson, and H. Yanagisawa Altered vascular remodeling in fibulin-5-deficient mice reveals a role of fibulin-5 in smooth muscle cell proliferation and migration PNAS, February 22, 2005; 102(8): 2946 - 2951. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. W. Fischer, S. A. Steitz, P. Y. Johnson, A. Burke, F. Kolodgie, R. Virmani, C. Giachelli, and T. N. Wight Decorin Promotes Aortic Smooth Muscle Cell Calcification and Colocalizes to Calcified Regions in Human Atherosclerotic Lesions Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2391 - 2396. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Farb, F. D. Kolodgie, J.-Y. Hwang, A. P. Burke, K. Tefera, D. K. Weber, T. N. Wight, and R. Virmani Extracellular Matrix Changes in Stented Human Coronary Arteries Circulation, August 24, 2004; 110(8): 940 - 947. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Shimizu-Hirota, H. Sasamura, M. Kuroda, E. Kobayashi, M. Hayashi, and T. Saruta Extracellular Matrix Glycoprotein Biglycan Enhances Vascular Smooth Muscle Cell Proliferation and Migration Circ. Res., April 30, 2004; 94(8): 1067 - 1074. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Jarvelainen, R. B. Vernon, M. D. Gooden, A. Francki, S. Lara, P. Y. Johnson, M. G. Kinsella, E. H. Sage, and T. N. Wight Overexpression of Decorin by Rat Arterial Smooth Muscle Cells Enhances Contraction of Type I Collagen In Vitro Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 67 - 72. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Nili, A. N. Cheema, F. J. Giordano, A. W. Barolet, S. Babaei, R. Hickey, M. R. Eskandarian, M. Smeets, J. Butany, G. Pasterkamp, et al. Decorin Inhibition of PDGF-Stimulated Vascular Smooth Muscle Cell Function: Potential Mechanism for Inhibition of Intimal Hyperplasia after Balloon Angioplasty Am. J. Pathol., September 1, 2003; 163(3): 869 - 878. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. J. Merrilees, J. M. Lemire, J. W. Fischer, M. G. Kinsella, K. R. Braun, A. W. Clowes, and T. N. Wight Retrovirally Mediated Overexpression of Versican V3 by Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation In Vitro and In Neointima After Vascular Injury Circ. Res., March 8, 2002; 90(4): 481 - 487. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Kolb, P. J. Margetts, P. J. Sime, and J. Gauldie Proteoglycans decorin and biglycan differentially modulate TGF-{beta}-mediated fibrotic responses in the lung Am J Physiol Lung Cell Mol Physiol, June 1, 2001; 280(6): L1327 - L1334. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. W. Fischer, M. G. Kinsella, B. Levkau, A. W. Clowes, and T. N. Wight Retroviral Overexpression of Decorin Differentially Affects the Response of Arterial Smooth Muscle Cells to Growth Factors Arterioscler. Thromb. Vasc. Biol., May 1, 2001; 21(5): 777 - 784. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. KOLB, P. J. MARGETTS, T. GALT, P. J. SIME, Z. XING, M. SCHMIDT, and J. GAULDIE Transient Transgene Expression of Decorin in the Lung Reduces the Fibrotic Response to Bleomycin Am. J. Respir. Crit. Care Med., March 1, 2001; 163(3): 770 - 777. [Abstract] [Full Text] [PDF] |
||||