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Circulation Research. 2000;86:676-683

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(Circulation Research. 2000;86:676.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Local Expression of Bovine Decorin by Cell-Mediated Gene Transfer Reduces Neointimal Formation After Balloon Injury in Rats

Jens W. Fischer, Michael G. Kinsella, Monika M. Clowes1, Stephanie Lara, Alexander W. Clowes, Thomas N. Wight

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
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract—Decorin is an extracellular matrix (ECM) proteoglycan that may modify vascular smooth muscle cell (SMC) function by altering the response to growth factors and the accumulation of ECM proteins during vascular injury. To investigate these possibilities in vivo, decorin was overexpressed at the site of arterial injury by cell-mediated gene transfer. Fischer rat SMCs were transduced in vitro with a retroviral construct that contained the bovine decorin gene and were subsequently seeded into injured rat carotid arteries. A species-specific antibody to bovine decorin and polymerase chain reaction primers were used to detect bovine decorin and distinguish it from endogenous rat decorin. Immunohistochemical and Northern analyses of rat carotid arteries revealed only low levels of rat decorin expression up to 8 weeks after balloon injury. However, after cell-mediated transfer of bovine decorin, strong expression of bovine decorin was verified by immunohistochemistry and reverse transcriptase–polymerase chain reaction. Four weeks after injury, the intimal area in vessels seeded with bovine decorin–overexpressing SMCs was significantly reduced by 35±4% (mean±SEM, n=9; P<0.01). Decorin overexpression also induced a higher intimal nuclear density and decreased volume of ECM. Specifically, immunostaining for versican and fibronectin was markedly reduced. In contrast, immunostaining for collagen type I was increased, and electron microscopy confirmed that collagen accumulation was altered. Bromodeoxyuridine labeling indicated that intimal SMC proliferation was not affected by the expression of bovine decorin. In summary, we demonstrate that gene transfer of the ECM proteoglycan, decorin, into the injured arterial wall reduces intimal ECM volume and alters the composition of the ECM.


Key Words: proteoglycans • smooth muscle cells • extracellular matrix • gene therapy • hyperplasia


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Intimal hyperplasia and subsequent arterial stenosis frequently reduce the long-term benefits of therapeutic vascular interventions in patients. The mechanisms leading to the development of neointimal hyperplasia in response to arterial injury involve increased migration, proliferation, and synthesis of the extracellular matrix (ECM) by smooth muscle cells (SMCs).1 2 Increased migration and proliferation of SMCs, which occur early after injury, are responsible for the formation of a highly cellular intimal lesion. The intimal lesion continues to expand after cell migration and proliferation cease as a result of ECM synthesis and accumulation. The majority of efforts to inhibit intimal hyperplasia have focused on inhibition of SMC proliferation and migration. However, inhibition of ECM accumulation could also be an effective strategy because the ECM constitutes {approx}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-ß1–induced 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
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Construction of the Bovine Decorin Retrovirus (LDSN)
The cDNA of bovine decorin (PG28, courtesy of Dr Marian Young, National Institute of Dental Research, Bethesda, Md) was inserted into the retroviral vector LXSN (courtesy of Dr A.D. Miller, Fred Hutchinson Cancer Research Center, Seattle, Wash),19 which was transfected into a packaging cell, and the resultant virus (LDSN) then used to transduce aortic SMCs from Fischer 344 rats as described previously.19 20 21

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 Masson’s trichrome procedure.23

Reverse Transcription–Polymerase 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 {alpha}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'-({alpha}-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
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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Low Expression of Decorin in the Intima of Rat Carotid Arteries After Balloon Injury
Balloon injury of left carotid arteries of male Fischer 344 rats was performed, and the animals were euthanized after 1, 2, 4, and 8 weeks. These time periods were chosen because they allowed us to examine the intima at the end of the proliferative and migratory phase (1 and 2 weeks) as well as during the phase of active ECM accumulation (2 to 4 weeks)1 2 during the response of the vessels to injury. Immunostaining for rat decorin with use of the polyclonal antibody LF-113 (Figure 1aDown) demonstrated that endogenous decorin is predominantly located in the adventitia of uninjured carotid arteries of Fischer 344 rats. After injury, decorin remains in the adventitia with only modest accumulation in the media or intima (Figure 1bDown to 1d). Even 8 weeks after balloon injury, decorin accumulation was low in the media and adventitia (not shown). In addition, Northern analysis of decorin mRNA expression was performed at 1, 2, and 4 weeks after injury. For this purpose, the carotid arteries of 3 rats were pooled, and the RNA was isolated. The autoradiograph of the labeled Northern blot and the image of the ethidium bromide–stained RNA gel were scanned and quantified by using NIH image software. The amount of rat decorin mRNA was expressed as the ratio between the signal for rat decorin mRNA and the ethidium bromide–stained rRNA. This procedure resulted in a value for rat decorin expression of 2.2 at 1 week, 2.6 at 2 weeks, and 1.6 at 4 weeks after balloon injury and 2.5 for the uninjured contralateral vessels. Therefore, the present data suggest that decorin is not upregulated during the migratory and proliferative response to injury, nor does it accumulate in the intima at later stages (up to 8 weeks).



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  		Figure 1. Decorin expression in balloon-injured arteries. Cross sections of carotid arteries were stained with anti-rat decorin antibody (LF-113, 1:1000). The arrows mark the external and internal elastic lamina; a indicates adventitia; m, media; and l, lumen. a, Uninjured carotid artery. b, One week after balloon injury. c, Two weeks after balloon injury. d, Four weeks after balloon injury. Bar=50 µm.

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 2BDown 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 2CDown) with a species-specific antibody to bovine decorin (LF-94).



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  		Figure 2. A, Schematic depiction of the retroviral vectors LXSN and LDSN. LTR indicates retroviral long terminal repeat; NEO, neomycin phosphotransferase; SV40, SV40 fragment containing early promoter; and pA, polyadenylation site. Arrows indicate transcriptional start sites and direction of transcription. B, Northern blot of mRNA from transduced Fischer rat SMCs. Blot was probed with a full-length bovine decorin cDNA. Lanes are as follows: 1, Fischer rat SMC transduced with the empty LXSN vector; 2, Fischer rat SMC transduced with LDSN. C, Expression of bovine decorin core protein by transduced Fischer rat SMCs. Conditioned medium of cultured LXSN- and LDSN-transduced cells was analyzed for bovine decorin core protein by use of a species-specific antibody (LF94) by Western blotting. Lanes are as follows: 1, buffer control; 2, LXSN-transduced SMC; and 3, LDSN-transduced SMC.

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 3aDown). As expected, no staining was observed after the same section was immunostained with the antibody to bovine decorin, LF-94 (Figure 3bDown). Similar results were obtained at 1 and 2 weeks after seeding of LXSN cells (not shown).



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  		Figure 3. Decorin expression in carotid arteries at 4 weeks after balloon injury and LXSN cell seeding. i indicates intima. a, Stained for rat decorin (LF113, 1:1000). b, Stained for bovine decorin (LF-94, 1:1000). Bar=50 µm.

The antibody LF-94 to bovine decorin enabled us to discriminate between endogenous rat decorin and the newly introduced bovine decorin. Figure 4ADown 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 transcription–polymerase 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 4CDown, 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 4BDown). Rat decorin is confined to the adventitia and does not change in the LDSN cell–seeded carotid arteries.



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  		Figure 4. Long-term (4-week) expression of bovine decorin (A) and rat decorin (B) in balloon-injured carotid arteries seeded with LDSN cells. a and d, One week. b and e, Two weeks. c and f, Four weeks. The arrows indicate the internal and the external elastic lamina. Bar=50 µm. C, Reverse transcription–PCR for bovine decorin mRNA at 4 weeks after seeding of LDSN cells. Lanes are as follows: 1, normal carotid artery; 2, LXSN cell–seeded vessel; 3, LDSN cell–seeded vessel; 4, bovine decorin cDNA; and right lane, molecular weight standard.

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 5ADown). 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 cell–seeded intimas versus 12.8±1% in LDSN cell–seeded intimas (mean±SEM, n=5). At 2 weeks, the percentage of BrdU-positive cells was 3.0±0.78% in LXSN cell–seeded intimas versus 4.3±0.9% in LDSN cell–seeded intimas (mean±SEM, n=5). These data suggest that intimal SMC proliferation was not affected by overexpression of decorin.



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  		Figure 5. Morphometric analysis of LXSN– and LDSN cell–seeded carotid arteries at 2 and 4 weeks. A, Intimal areas are reduced by 35±4% in LDSN cell–seeded vessels at 4 weeks (P<0.01). B, Nuclear density is significantly increased in decorin-overexpressing intimas at 4 weeks compared with the LXSN cell–seeded intimas (P<0.01). C, Total number of nuclei is not altered by overexpression of decorin at 2 weeks or 4 weeks. Shown are the mean±SEM at 2 weeks (n=5) and at 4 weeks (n=9).

The intimal nuclear density was determined by counting SMC nuclei per high-power field. As shown in Figure 5BUp, intimal nuclear density was significantly (P<0.01) higher in LDSN cell–seeded vessels at 4 weeks compared with LXSN cell–seeded intimas, suggesting that ECM accumulation might be reduced in decorin-overexpressing intimas. The observations that the total numbers of nuclei (Figure 5CUp) were the same in the intimas of LXSN cell– and LDSN cell–seeded vessels but that the intimal area was reduced in LDSN cell–seeded 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 cell–seeded carotid arteries was analyzed by immunostaining. Four weeks after cell seeding, the staining for versican and fibronectin was much less in the LDSN cell–seeded vessels (Figures 6gDown and 6hDown) than in the LXSN cell–seeded vessels (Figures 6cDown and 6dDown). On the other hand, the LDSN cell–seeded intimas showed more pronounced staining for collagen, as shown by immunohistochemistry for collagen type I (Figure 6fDown) and by histochemistry using Masson’s trichrome stain (Figure 7bDown). 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 8ADown and 8BDown), whereas fewer collagen fibrils were present in the LXSN–seeded vessels (Figures 8CDown and 8DDown). 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 8CDown and 8DDown). 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 8ADown and 8BDown). 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



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  		Figure 6. Immunostaining for bovine decorin (LF94, 1:1000), collagen type I (LF67, 1:1000), versican (1:100), and fibronectin (1:4000) in LXSN cell–seeded (a through d) and LDSN cell–seeded (e through h) carotid arteries at 4 weeks after seeding. a and e, Bovine decorin. b and f, Collagen type I. c and g, Versican. d and h, Fibronectin. The arrows mark the internal and external elastic lamina. Bar=50 µm.



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  		Figure 7. Masson’s trichrome stain of LXSN cell–seeded (a) and LDSN cell–seeded (b) vessels 4 weeks after seeding. Bar=50 µm.



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  		Figure 8. Representative electron micrographs from the deeper layers of the thickened intimas seeded with decorin-overexpressing SMCs (A and B) and seeded with vector control–transduced cells (C and D). A, Cross sections of collagen fibrils (c) fill the entire ECM in the decorin-seeded intimas. Small immature elastic fibers (arrowheads) are frequently seen associated with the surface of the SMCs. Original magnification x5000. Bar=1 µm. B, High-magnification micrograph of the ECM shown in panel A demonstrates an extensive collagen network surrounding the SMC and immature elastin fibers associated with the cell surface. Original magnification x20 000. Bar=200 nm. C, ECM in the vector control–seeded intimas has large areas of amorphous material and occasional bundles of collagen fibrils, either cut in cross section (c) or longitudinal section. Original magnification x5000. Bar=1 µm. D, High-magnification micrograph of the ECM shown in panel C demonstrates the loose amorphous ECM material and occasional longitudinal sections of a few collagen fibrils. Original magnification x20 000. Bar=200 nm.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, decorin was overexpressed locally in balloon-injured carotid arteries, and the effect of this proteoglycan on intimal hyperplasia, intimal ECM accumulation, and composition was examined. It is of interest that although vascular injury causes increases in a variety of ECM molecules, including several proteoglycans,27 28 29 endogenous decorin deposition does not increase, as shown in the present study. The absence of a decorin response after balloon injury makes expression of this proteoglycan a reasonable choice for evaluating the effect of this molecule on the neointimal response.

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 cell–seeded and decorin cell–seeded 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 cell–seeded 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-ß1–independent 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-1–stimulated 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
 
This study was supported by National Institutes of Health grants (HL-18645 and HL-52459) and a postdoctoral fellowship from the Ernst Schering Research Foundation, Berlin, Germany, to Dr Jens W. Fischer. We thank Holly Lea for assistance with the surgical procedures. This article is dedicated to the memory of Monika Meyer Clowes.


*    Footnotes
 
1 Deceased. Back

Received July 12, 1999; accepted December 30, 1999.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
  2. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208–215.[Medline] [Order article via Infotrieve]
  3. Thyberg J, Hultgardh-Nilsson A. Fibronectin and the basement membrane components laminin and collagen type IV influence the phenotypic properties of subcultured rat aortic smooth muscle cells differently. Cell Tissue Res. 1994;276:263–271.[Medline] [Order article via Infotrieve]
  4. Thyberg J, Blomgren K, Roy J, Tran PK, Hedin U. Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin. J Histochem Cytochem. 1997;45:837–846.[Abstract/Free Full Text]
  5. Koyama H, Raines EW, Bornfeldt KE, Roberts JM, Ross R. Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of Cdk2 inhibitors. Cell. 1996;87:1069–1078.[Medline] [Order article via Infotrieve]
  6. Bauters C, Marotte F, Hamon M, Oliviero P, Farhadian F, Robert V, Samuel JL, Rappaport L. Accumulation of fetal fibronectin mRNAs after balloon denudation of rabbit arteries. Circulation. 1995;92:904–911.[Abstract/Free Full Text]
  7. Krusius T, Ruoslahti E. Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci U S A. 1986;83:7683–7687.[Abstract/Free Full Text]
  8. Day AA, McQuillan CI, Termine JD, Young MR. Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone. Biochem J. 1987;248:801–805.[Medline] [Order article via Infotrieve]
  9. Iozzo RV. The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth. Crit Rev Biochem Mol Biol. 1997;32:141–174.[Medline] [Order article via Infotrieve]
  10. Schonherr E, Broszat M, Brandan E, Bruckner P, Kresse H. Decorin core protein fragment Leu155-Val260 interacts with TGF-ß but does not compete for decorin binding to type I collagen. Arch Biochem Biophys. 1998;355:241–248.[Medline] [Order article via Infotrieve]
  11. Border WA, Noble NA, Yamamoto T, Harper JR, Yamaguchi Y, Pierschbacher MD, Ruoslahti E. Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease. Nature. 1992;360:361–364.[Medline] [Order article via Infotrieve]
  12. Giri SN, Hyde DM, Braun RK, Gaarde W, Harper JR, Pierschbacher MD. Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis. Biochem Pharmacol. 1997;54:1205–1216.[Medline] [Order article via Infotrieve]
  13. Yamaguchi Y, Ruoslahti E. Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation. Nature. 1988;336:244–246.[Medline] [Order article via Infotrieve]
  14. Patel S, Santra M, McQuillan DJ, Iozzo RV, Thomas AP. Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells. J Biol Chem. 1998;273:3121–3124.[Abstract/Free Full Text]
  15. De Luca A, Santra M, Baldi A, Giordano A, Iozzo RV. Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases. J Biol Chem. 1996;271:18961–18965.[Abstract/Free Full Text]
  16. Coppock DL, Kopman C, Scandalis S, Gilleran S. Preferential gene expression in quiescent human lung fibroblasts. Cell Growth Differ. 1993;4:483–493.[Abstract]
  17. Kuc IM, Scott PG. Increased diameters of collagen fibrils precipitated in vitro in the presence of decorin from various connective tissues. Connect Tissue Res. 1997;36:287–296.[Medline] [Order article via Infotrieve]
  18. Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol. 1997;136:729–743.[Abstract/Free Full Text]
  19. Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989;7:980–982, 984–986, 989–990.[Medline] [Order article via Infotrieve]
  20. Clowes MM, Lynch CM, Miller AD, Miller DG, Osborne WR, Clowes AW. Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes. J Clin Invest. 1994;93:644–651.
  21. Corsaro CM, Pearson ML. Enhancing the efficiency of DNA-mediated gene transfer in mammalian cells. Somat Cell Genet. 1981;7:603–616.
  22. Forough R, Koyama N, Hasenstab D, Lea H, Clowes M, Nikkari ST, Clowes AW. Overexpression of tissue inhibitor of matrix metalloproteinase-1 inhibits vascular smooth muscle cell functions in vitro and in vivo. Circ Res. 1996;79:812–820.[Abstract/Free Full Text]
  23. Masson. Trichrome stainings and their preliminary technique. J Tech Methods. 1929;12:75–90.
  24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
  25. Jarvelainen HT, Kinsella MG, Wight TN, Sandell LJ. Differential expression of small chondroitin/dermatan sulfate proteoglycans, PG-I/biglycan and PG-II/decorin, by vascular smooth muscle and endothelial cells in culture. J Biol Chem. 1991;266:23274–23281.[Abstract/Free Full Text]
  26. Lynch CM, Clowes MM, Osborne WR, Clowes AW, Miller AD. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy. Proc Natl Acad Sci U S A. 1992;89:1138–1142.[Abstract/Free Full Text]
  27. Strauss BH, Chisholm RJ, Keeley FW, Gotlieb AI, Logan RA, Armstrong PW. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ Res. 1994;75:650–658.[Abstract/Free Full Text]
  28. Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am J Pathol. 1994;144:1348–1356.[Abstract]
  29. Karim MA, Miller DD, Farrar MA, Eleftheriades E, Reddy BH, Breland CM, Samarel AM. Histomorphometric and biochemical correlates of arterial procollagen gene expression during vascular repair after experimental angioplasty [see Comments]. Circulation. 1995;91:2049–2057.[Abstract/Free Full Text]
  30. Hausser H, Groning A, Hasilik A, Schonherr E, Kresse H. Selective inactivity of TGF-ß/decorin complexes. FEBS Lett. 1994;353:243–245.[Medline] [Order article via Infotrieve]
  31. Yamaguchi Y, Mann DM, Ruoslahti E. Negative regulation of transforming growth factor-beta by the proteoglycan decorin. Nature. 1990;346:281–284.[Medline] [Order article via Infotrieve]
  32. Isaka Y, Brees DK, Ikegaya K, Kaneda Y, Imai E, Noble NA, Border WA. Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney. Nat Med. 1996;2:418–423.[Medline] [Order article via Infotrieve]
  33. Border WA, Ruoslahti E. Transforming growth factor-beta in disease: the dark side of tissue repair. J Clin Invest. 1992;90:1–7.
  34. Rasmussen LM, Wolf YG, Ruoslahti E. Vascular smooth muscle cells from injured rat aortas display elevated matrix production associated with transforming growth factor-beta activity. Am J Pathol. 1995;147:1041–1048.[Abstract]
  35. Noble NA, Harper JR, Border WA. In vivo interactions of TGF-ß and extracellular matrix. Prog Growth Factor Res. 1992;4:369–382.[Medline] [Order article via Infotrieve]
  36. Grande JP. Role of transforming growth factor-beta in tissue injury and repair. Proc Soc Exp Biol Med. 1997;214:27–40.[Abstract]
  37. Nabel EG, Shum L, Pompili VJ, Yang ZY, San H, Shu HB, Liptay S, Gold L, Gordon D, Derynck R, Nabel GJ. Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A. 1993;90:10759–10763.[Abstract/Free Full Text]
  38. Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model. J Clin Invest. 1994;93:1172–1178.
  39. Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E. Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J. 1994;302:527–534.
  40. Schlumberger W, Thie M, Rauterberg J, Robenek H. Collagen synthesis in cultured aortic smooth muscle cells: modulation by collagen lattice culture, transforming growth factor-ß1, and epidermal growth factor. Arterioscler Thromb. 1991;11:1660–1666.[Abstract/Free Full Text]
  41. Brown DC, Vogel KG. Characteristics of the in vitro interaction of a small proteoglycan (PG II) of bovine tendon with type I collagen. Matrix. 1989;9:468–478.[Medline] [Order article via Infotrieve]
  42. Vogel KG, Paulsson, M., Heinegård D. Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J. 1984;223:587–597.[Medline] [Order article via Infotrieve]
  43. Pins GD, Christiansen DL, Patel R, Silver FH. Self-assembly of collagen fibers: influence of fibrillar alignment and decorin on mechanical properties. Biophys J. 1997;73:2164–2172.[Abstract/Free Full Text]
  44. Scott JE. Extracellular matrix, supramolecular organisation and shape. J Anat. 1995;187:259–269.
  45. Pringle GA, Dodd CM. Immunoelectron microscopic localization of the core protein of decorin near the d and e bands of tendon collagen fibrils by use of monoclonal antibodies. J Histochem Cytochem. 1990;38:1405–1411.[Abstract]
  46. Evanko SP, Vogel KG. Ultrastructure and proteoglycan composition in the developing fibrocartilaginous region of bovine tendon. Matrix. 1990;10:420–436.[Medline] [Order article via Infotrieve]
  47. Sztrolovics R, White RJ, Poole AR, Mort JS, Roughley PJ. Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism. Biochem J. 1999;339:571–577.
  48. Lin H, Wilson JE, Roberts CR, Horley KJ, Winters GL, Costanzo MR, McManus BM. Biglycan, decorin and versican protein expression patterns in coronary arteriopathy of human cardiac allograft: distinctness as compared to native atherosclerosis. J Heart Lung Transplant. 1996;15:1233–1247.[Medline] [Order article via Infotrieve]
  49. Radhakrishnamurthy B, Tracy RE, Dalferes ER Jr, Berenson GS. Proteoglycans in human coronary arteriosclerotic lesions. Exp Mol Pathol. 1998;65:1–8.[Medline] [Order article via Infotrieve]
  50. Riessen R, Isner JM, Blessing E, Loushin C, Nikol S, Wight TN. Regional differences in the distribution of the proteoglycans biglycan and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries. Am J Pathol. 1994;144:962–974.[Abstract]
  51. Evanko SP, Raines EW, Ross R, Gold LI, Wight TN. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor-beta. Am J Pathol. 1998;152:533–546.[Abstract]
  52. Snow AD, Bolender RP, Wight TN, Clowes AW. Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells. Am J Pathol. 1990;137:313–330.[Abstract]
  53. Clark RAF. Wound repair: overview and general considerations. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. 2nd ed. New York, NY: Plenum Press; 1996:3–35.
  54. Aikawa M, Rabkin E, Okada Y, Voglic SJ, Clinton SK, Brinckerhoff CE, Sukhova GK, Libby P. Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma. Circulation. 1998;97:2433–2444.[Abstract/Free Full Text]
  55. Rekhter MD. Collagen synthesis in atherosclerosis: too much and not enough. Cardiovasc Res. 1999;41:376–384.[Abstract/Free Full Text]



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