Matrix Genes after Arterial Injury - Europe PMC

American Journal of Pathology, Vol. 144, No. 6, June 1994 CopynIght © American Society for Investigative Pathology

Smooth Muscle Cell Expression of Extracellular Matrix Genes after Arterial Injury

Seppo T. Nikkari,* Hannu T. J&rvelainen,t Thomas N. Wight,t Marina Ferguson,t and Alexander W. Clowes* From the Departments of Surgery* and Pathology,t University of Washington, Seattle, Washington

Accumulation of extraceUular matrix (ECM) after arterial injury is an important event in the development of intimal thickening and is modulated by heparin. To investigate the regulation of matrix protein expression, we have analyzed messenger RNA levels by Northern blotting for various ECM proteins in the rat carotid artery balloon injury modeL RNA was extracted from normal arteries and from intima-medial preparations at 2 days, I week, 2 weeks, and 4 weeks after baloon injury of arteries in animals receiving either saline or heparin infusion. Transcripts for the heparan sulfate proteoglycans perlecan, syndecan, and ryudocan, the chondroitin sulfate proteoglycan versican, the dermatan sulfate proteoglycan biglycan, type I procoUagen, and tropoelastin aU were increased on Northern blots beginning at I week after injury. By in situ hybridization, the transcripts for elastin and biglycan were primarily localized to smooth muscle ceUs in the intima and were diminished by heparin in proportion to the decrease in intimal mass. Other matrix genes (perlecan, ryudocan) were expressed in the intima and media and were not affected by heparin. The results support the conclusion that ECM gene expression is a relatively late event in the response of the carotid artery, and that some of the genes are expressed only in the intima whereas others are expressed in both the intima and media. (AmJ Pathol 1994, 144:1348-1356)

The intimal hyperplastic response of smooth muscle cells (SMCs) to arterial reconstruction is the primary cause of late restenosis in humans.1 The extracellular matrix (ECM) is a major component of these lesions. For example, at anastomoses of arteriovenous shunts used for chronic hemodialysis in humans, intimal hy-

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perplastic lesions form and contain collagen and elastin in greatest concentration deep in the intima, whereas close to the lumen most of the extracellular volume consists of proteoglycan.2 The ballooned rat carotid artery has been used to study the intimal hyperplastic response. In this model the endothelium is removed and about 25% of the medial SMCs are damaged by the intraluminal passage of an inflated balloon catheter.3 The remaining medial SMCs start proliferating 1 day after injury. Migrating medial SMCs begin to form the neointima after 4 days, and these cells continue to proliferate in the intima up to about 2 weeks. At later stages, although the SMC content of the wall does not change, the intima is further thickened by the accumulation of large amounts of ECM including elastin, collagen, and proteoglycan. It is estimated that at 3 months after injury the ECM makes up 80% of the lesion.3 The expression of ECM protein genes and the accumulation of ECM have an important role in the pathogenesis of intimal lesions. Not only do the ECM proteins occupy space but they may also modify other biological functions in the artery wall.4 The accumulation of ECM proteins can also be regulated. For example, the glycosaminoglycan heparin is known to inhibit both SMC migration and proliferation,5 and it also decreases the levels of collagen and elastin protein and increases chondroitin sulfate proteoglycan (CSPG) and heparan sulfate proteoglycan (HSPG) in the neointima.6 The purpose of the present study was to assess the messenger RNA (mRNA) expression of certain matrix macromolecules at various times after balloon injury and to determine the effect of heparin administration on this response. All of the studied ECM protein transcripts were induced by injury, and our findings suggest that accumulation of ECM after experimental arterial injury may partly be regulated at the mRNA Supported by grants from the Pirkanmaa Regional Fund under the auspices of the Finnish Cultural Foundation and by NIH grant HL1 8645. Accepted for publication January 27, 1994. Address reprint requests to Dr. Alexander W. Clowes RF-25, Department of Surgery, University of Washington, Seattle, WA 98195.

SMC Expression of ECM

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level. The present results also indicate that the effect of heparin on ECM protein accumulation cannot be accounted for by a change in mRNA level.

Materials and Methods Arterial Injury Model Five-month-old male Sprague-Dawley rats (weighing 350 to 450 g) were anesthetized and subjected to bilateral common carotid artery balloon catheter injury as previously described.7 Both common carotid arteries were removed from normal animals or at 2 days, 1 week, 2 weeks, and 4 weeks after injury, stripped of adventitial tissue, and snap-frozen in liquid nitrogen. Approximately 6 to 10 arteries were used for each time point, and the experiment was repeated three times. Twenty-seven rats were perfusion-fixed with 10% formalin, and morphological sections from the middle of the common carotid arteries were obtained after paraffin embedding. Four-week, 2-ml miniosmotic pumps (0.5 pl/hour) (Alzet, Alza Corp., Palo Alto, CA) were placed in the subcutaneous tissue over the backs of the animals and connected to Silastic catheters inserted into the left jugular veins. These pumps contained normal saline with or without heparin (0.3 mg/kg/hour i.v.) (Choay 1772, Choay Recherche, Sanofi Inc., Paris, France).5

RNA Isolation and Analysis The frozen carotid tissue was crushed to a fine powder on dry ice, and total RNA was prepared according to the method of Chomczynski and Sacchi.8 Amounts of RNA were determined by spectrophotometric absorbance at 260 nm. For Northern blot analysis, 15 pg of purified total RNA per lane was electrophoresed on 1.2% denaturing formaldehyde agarose gels, transferred to nylon membranes (Zeta Probe; Bio-Rad Laboratories, Richmond, CA), and cross-linked to the membranes with short-wave ultraviolet light. Serially diluted concentrations of total RNA from one experiment were applied to nitrocellulose membranes by a vacuum manifold apparatus, followed by baking at 80 C for 2 hours. The prehybridizing and hybridizing solutions for the nylon membranes contained 50% formamide in 0.14 M sodium phosphate buffer, pH 7.2, with 0.25 M NaCI, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 10% polyethylene glycol. For the nitrocellulose membranes, the prehybridizing and hybridizing solution contained 50% formamide, 5X Denhardt's solution, 6X SSPE, and 0.5% SDS. Complementary DNA (cDNA) probes were labeled with [32P]dCTP by nick translation and added to the

hybridizing solutions to a specific activity of 2 x 106 cpm/ml. Hybridization was at 42 C for 15 to 20 hours. The blots were then washed several times; the final wash was in 0.1X standard sodium citrate (SSC) and 0.1% SDS at 65 C. Quantitation was performed on a Phosphorlmager (Markey Molecular Medicine Center, University of Washington, Seattle, WA). Exposures were done on Kodak XAR-2 film (Eastman Kodak Co., Rochester, NY) at -70 C. Some autoradiograms were also scanned with a gel scanner (Hoefer Scientific Instruments, San Francisco, CA). All loadings were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPD). Results were expressed as fold increase above value obtained from uninjured carotids. cDNA probes were as follows: tropoelastin, a 0.9-kb rat cDNA,9 was generously provided by Dr. C. D. Boyd; ryudocan (syndecan-4), a 0.2-kb rat cDNA, and syndecan (syndecan-1), a 0.6-kb rat cDNA,10 were a kind gift of Drs. N. Shworak and R. Rosenberg; perlecan, a 1.1-kb human cDNA,11 was a generous gift of Dr. R. lozzo; versican, a 3.5-kb cDNA (C7) encoding the middle portion, and a 1.7-kb cDNA (C10) encoding the carboxyl terminal portion of human versican, 12 were generously supplied by Dr. R. LeBaron; versican, a 1.5-kb rat cDNA showing 84% homology in the link region with human versican, was provided kindly by Dr. J. Lemire13 pro-a 1(1) collagen, a 1.4-kb human cDNA14: biglycan (PG-I), a human 1.7-kb cDNA15 was generously provided by Dr. L. Fisher; GAPD, a 1.2-kb human cDNA16 was from the American Type Culture Collection, Rockville, MD.

In Situ Hybridization Rats were perfusion-fixed with 10% formalin in 0.1 M phosphate buffer, pH 7.4, and further postfixed overnight in the same fixative. After a 1-day incubation in phosphate buffer alone, the rings were dehydrated and embedded in paraffin. Five-p cross-sections were deparaffinized and dehydrated in graded alcohols, rinsed in 0.5X SSC, and incubated with 5 pg/ml proteinase K (Sigma Chemical Co., St. Louis, MO) for 30 minutes at 37 C. The slides were then washed briefly in 0.5X SSC and prehybridized in 100 pl of hybridization buffer (50% formamide, 0.3 M NaCI, 20 mM Tris, pH 8.0, 5 mM EDTA, 1X Denhardt's, 10% dextran sulfate, and 10 mM dithiothreitol). The prehybridization was performed in airtight boxes containing blotting paper saturated with 50% formamide and 4X SSC on the bottom at 50 C for 2 hours. 35S-labeled riboprobes were generated as previously described.17'18 Riboprobes were added to the prehybridized slides in 25 p1 of fresh hybridizing solution at 3.5 x 105 cpm per slide. Hybridization was

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continued overnight at 50 C. The slides were subsequently rinsed three times in 0.5X SSC and immersed in RNAse A solution (20 pg/ml in 0.5 M NaCI and 10 mM Tris, pH 8.0) for 30 minutes at 37 C. The slides were rinsed in 2X SSC and washed for 2 hours in 0.1X SSC and 0.5% Tween 20 at 50 C or 37 C. After rinsing in 2X SSC, the slides were dipped in Kodak NTB-2 nuclear track emulsion and exposed at 4 C for 7 or 14 days.

Measurement of the Intimal Area Paraffin-embedded cross-sections from the middle of rat carotid arteries were deparaffinized in xylene and rehydrated in graded alcohol. Hematoxylin and eosin-stained sections were projected onto a computerized digitalizing pad with a camera lucida to measure the intimal area.

Results Intimal Area Balloon injury to the carotid arteries caused an inin intimal area over a period of 4 weeks (Figure 1). Intimal thickening was quantitatively less in heparin-treated rats as compared with saline-treated controls. The development of myointimal thickening at 1 week was completely inhibited by heparin (P < 0.05); inhibition at 2 weeks was 65% (P < 0.05), and at 4 weeks 28% (ns). crease

*

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saline

heparin

0.30-

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to U-

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0.10-

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ECM Gene Expression We measured relative steady-state levels for ECM mRNAs extracted from intima medias of ballooninjured rat carotid arteries. Uninjured carotids served as controls, and their transcript levels were given a relative unit value of 1. Transcripts for all genes were detected in uninjured (control) arteries and increased at various times after injury (Figure 2). Perlecan transcripts increased at 2 weeks in injured carotids. This increase persisted at 4 weeks. The steady-state levels of the syndecan transcript doublet10 increased gradually, peaking at 2 weeks. Ryudocan, a novel member of the syndecan family of transmembrane HSPGs,10 increased gradually after injury beginning at 2 days. In our model of intimal hyperplastic response there was an increase of the polydisperse versican transcripts first at 2 weeks; this induction persisted at 4 weeks. Similar results were obtained with a mixture of the two human cDNAs as with the rat cDNA probe (data not shown). We found that biglycan transcripts were already abundant in control rat carotids and showed an induction between 2 and 4 weeks after injury. Type procollagen mRNA levels showed an initial decrease at 2 days. Both tropoelastin and type procollagen transcript levels peaked at 2 weeks, then diminished at 4 weeks.

The Effect of Heparin on ECM Gene Expression Heparin treatment appeared to decrease significantly transcript levels of tropoelastin and biglycan genes in the total intima-medial preparations at 2 weeks after injury, compared with saline treatment (Table 1). This decrease was less apparent at 4 weeks for the same transcripts, suggesting that these genes were induced in the intima, and that the difference between heparin and saline treatment was secondary to intimal thickness. In contrast, steady-state mRNA levels for type collagen, perlecan, ryudocan, syndecan, and versican did not significantly differ between heparinand saline-treated rats at any time point.

-

-9p-

1 wk

2 wk

-1

4 wk

Figure 1. Intimal tbickening in balloon-injured carotid arteries of rats infused with either saline or beparin. The carotids were barvested after 1 week, 2 weeks, and 4 weeks, perfusion-fixed with 10% formaldebyde, sectioned, and stained: the intimal areas were measured with a digitalplanimeter. Values are the mean area + SE (three animalsltime pointlexperiment). Heparin significantly inhibited myointimal thickening compared with saline at 1 week and 2 weeks by Mann-Whitney U test (P < 0.05); at 4 weeks the difference was not statistically significant.

In Situ Hybridization Because the largest decrease in gene expression in the heparin-treated rat carotid arteries was for tropoelastin and biglycan at 2 weeks after injury, we used in situ hybridization to localize the induction of their transcripts. Our results showed that there was a small amount of tropoelastin mRNA in medial SMCs


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Injury

Cont 2wk 4wk 2.0 ryudocan

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1.5

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Cont 2d

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versican

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-

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2 type I collagen

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Figure 2. Northern analysis of increase in FGCM mRNA levels after carotid injury. RNA was harvestedfrom inlured carotids after 2 day.s, 1 week, 2 weeks, and 4 weeks; uninjuired carotids were used as controls. 15 pg of RNA for each time point wvas hybridized to spec ific cDNA probe,s. The ressults were quantified by^ PhosphorImager autoradiography and expressed as relative radioactivity units. All results wvere niormalized to GAPD. The data represents the means of three different experinents (+SE); three to five animals/time pointlexperinient.

of uninjured vessels (Figure 3A); the levels were similar at 2 days after injury (Figure 3B). At 1 week there

increase of tropoelastin transcripts in some medial SMCs (Figure 3C). A few cells in the neointima

was an

at 1 week showed increased signal. Strong signal was observed at 2 weeks in virtually all neointimal cells (Figure 3D). The signal was equally strong in the intimal cells of heparin-treated animals at 2 weeks (Fig-

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Table 1. Effect of Heparin Administration on ECM mRNA Levels at 2 weeks after Injury*

ECM component Ryudocan Syndecan Versican Perlecan Biglycan Elastin Type collagen

Saline

Heparin

1.6 1.9 1.9 1.9 1.6 3.9 2.0

1.6 1.5 2.0 2.1

1.0t 2.3t 1.2

* All results are normalized to GAPD and are expressed as fold inductions over uninjured carotid values (means of two to three different experiments). Three to five rats were pooled for each experiment. t Significantly different from saline group by Mann-Whitney U test (P 0.05).

ure 3E), even though the intimal thickness was decreased. By 4 weeks the induction of tropoelastin transcripts was confined predominantly to the SMCs nearest the lumen. Tropoelastin induction was also seen in some adventitial cells mostly at 1 and 2 weeks. The induction of biglycan at 2 weeks was also predominantly in the intima by in situ hybridization, although its transcripts were abundant already in the uninjured media (data not shown). Biglycan expression was not altered by heparin treatment. There were no differences between saline- and heparin-treated steady-state mRNA levels for several of the proteoglycans. We examined the localization of perlecan transcripts at 2 weeks after injury in the carotids of heparin- and saline-treated animals (Figure 4). Perlecan mRNA was almost undetectable in uninjured vessels (Figure 4A). At 2 weeks after injury the induction of perlecan transcripts was seen in both intima and media in saline-treated rats (Figure 4B). A similar induction was seen in heparin-treated animals (Figure 4C). Likewise, an intima-medial induction was also seen for ryudocan in saline- and heparin-treated rats (data not shown). Sense probe controls for tropoelastin and perlecan produced very low background signal (data not shown).

Discussion In the present study we demonstrate that all the studied ECM genes were induced over time after injury. The mRNA levels of cell-surface HSPGs syndecan and ryudocan increased gradually, while the other ECM transcripts increased after a lag period of about 1 week after injury. We did not study time points earlier than 2 days, but a previous study has shown that there is a down-regulation of transcripts for tropoelastin and type procollagen during this period.17 This pattern is different from what is observed for inductions of immediate-early genes such as thrombospondin and

c-myc in vivo.19 These immediate-early genes have been shown to undergo a biphasic induction peaking first at approximately 2 to 6 hours after balloon injury. We now show that ECM gene induction is maximal near the end of the proliferative phase of neointima formation at 2 weeks,3 after which some of these genes, like tropoelastin and type procollagen, show decline in transcripts. Perlecan is an interstitial matrix HSPG that has been isolated from the basement membranes of several cell types.11 It has also been shown by immunocytochemistry to be a component of the rat carotid neointima.20 We report a twofold increase in the transcripts of perlecan at 2 and 4 weeks after injury. We also demonstrate increases in the transcripts of the cell surface HSPGs ryudocan and syndecan. They both belong to the syndecan family of integral cell surface HSPGs, and their transcripts have been detected in rat aortic SMCs.10 Syndecan protein has also been demonstrated in vivo in aortic SMCs of neonatal rats and to a lesser extent in adult rats by immunocytochemistry.21 Syndecans bind to other ECM components, and they may also regulate effects of growth factors by acting as low-affinity receptors for mitogens such as basic fibroblast growth factor.22'23 Our in situ data indicate that perlecan and ryudocan are induced after injury in the media as well as in the neointima. It may be that, after injury in response to mitogens, SMCs first up-regulate cell surface HSPGs, which could play a role in cell replication and migration. The induction of other ECM HSPGs, such as perlecan after a lag period of 1 week might lead to sequestration of heparin-binding growth factors away from cell surface receptors, thus leading to SMC quiescence.23,24 Alternatively, these HSPGs might be involved in the organization of ECM structures such as basement membranes.4 Versican is a CSPG which was originally isolated from fibroblasts.12 Versican synthesis is stimulated in cell culture conditions by platelet-derived growth factor and transforming growth factor-P.25 Previous studies have shown that there is an accumulation of a large versican-like CSPG in the intimal lesions of atherosclerotic vessels,26 and staining with versican antibody is seen in both human aortic and coronary atherosclerotic intima (T.N. Wight, unpublished observations). CSPGs such as versican have been implicated in the entrapment of low-density lipoprotein in the artery wall during atherogenesis.27 We found that versican mRNA was increased at 2 and 4 weeks after injury, suggesting that there may be an accumulation of this PG in restenotic lesions. The biglycan PG has two dermatan sulfate chains. It is localized at cell surfaces and pericellularly.2a A


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-

-

:i.

m,

z

'0.,ll.* .'

-4

Figure 3. Darkfieldphotomicrographs showing time course of tropoelastin transcript induction after rat carotid artery injury by in situ hybnidization. A: Uninjured carotid. Injured carotids are shown at B: 2 days, C: 1 week, D: 2 weeks, E: 2 weeks with beparin treatment and F: 4 weeks after injuiry. The effect of heparin treatment is shown on/V at 2 weeks, because the inductions at the other time points did not significantly differfrom the saline group. Despite the difference in intimal thickness at 2 weeks in beparin versus saline-treated animaLs, intimal cells in both groups displayed equial cytoplasmic intensity of signal. At 4 weeks, tropoelastin induction was confined mainly to the cells closest to the lumen. Arrows shouw the internal elastic lamina. Lumen is at the top (x 400).

biglycan-type PG has been identified to be a major component of intima-medial preparations from human aorta,29 and it is ubiquitously expressed in the endothelium and all cell layers of human coronary atherosclerotic lesions (T.N. Wight, unpublished observations). The exact function of biglycan is unknown, but it has been found to inhibit transforming growth factor-4,30 which is the most potent inducer of ECM formation.31 Biglycan transcripts were abundant already in uninjured rat carotids, and the message was induced in the neointima after injury. However, because of the high medial baseline expression, biglycan transcripts do not appear to be a marker for neointimal cells. The major fibrous proteins of the vascular ECM are type collagen and elastin. They have been shown to be deposited in neointimal tissue after arterial injury,32

and their mRNA levels have recently been shown to be developmentally regulated.17 An abundance of elastic lamellae and collagen fibrils is seen in the rat neointima after balloon injury.6 We have previously demonstrated that fibrillar collagen is deposited preferentially in the lower neointima, whereas elastin is deposited more uniformly.6 In the present study there was a small decrease of type procollagen at 2 days possibly associated with medial SMC proliferation.3 A maximal increase of both type procollagen and tropoelastin mRNAs was seen at 2 weeks after injury. The transcript levels decreased from these levels at 4 weeks. It seems that expression of both type collagen and elastin is a highly controlled process, and overexpression ceases after the neointima has been established. The neointimal SMCs appeared to represent a different phenotypic population from the me-

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Figure 4. Darkfieldphotomicrographs showing localization ofperlecan transcnpt induction after rat carotid artery injury by in situ hybridization. A: uninjured carotid. B: 2 weeks after injury with saline treatment, and C: 2 weeks after injury with heparin treatment. Induction of transcnpts is in both intimal and medial cells in both groups. Arrows span the width of the media and point at the internal elastic lamina. Lumen is at the top ( x 800).

dial cells, at least in regard to expression of tropoelastin mRNA. Neointimal cells in culture also appear to have a different phenotype from medial cells and appear to resemble more closely SMCs derived from neonatal aorta. 17 At 4 weeks after injury this subpopulation seems to be confined to the periluminal surface of the neointima, whereas the underlying SMCs have turned down their tropoelastin mRNA expression. We have previously shown that the luminal cells are still cycling while the underlying SMCs have resumed the quiescent state.7 In the present study heparin inhibited neointima formation, and at 2 weeks it decreased transcript levels for tropoelastin and biglycan in total intima-medial extracts analyzed by Northern blotting. However, our in situ hybridization studies demonstrated that the neointimal cells in both saline- and heparin-treated animals were equally positive for tropoelastin. Inasmuch as the induction of tropoelastin occurred mainly in the intima, the decrease in transcript levels on Northern blots in the heparin group intima-medial extracts when compared with those of the saline group can be explained by the difference in intimal cell numbers. The inhibition by heparin of the induction of biglycan transcripts was also secondary to reduction of intimal thickness. This might also explain the relative decrease by heparin of type procollagen transcripts, given that the induction of this gene has previously been shown to occur mainly in the neointima. 17 All the other ECM gene transcripts studied by Northern blot-

ting were not significantly affected by heparin, and inductions of these genes (eg, perlecan and ryudocan) were found in both intima and media. Taken together, it seems that, even though heparin decreases neointimal thickening, it appears to have no direct effect on SMC expression of ECM transcripts. We have studied rat aortic SMCs in cell culture and determined that heparin does not affect mRNA levels of syndecan, ryudocan, perlecan, versican, biglycan, tropoelastin, type procollagen, or GAPD after serum stimulation (STN; unpublished observations). Our previous observations of increased accumulation of proteoglycans and a decrease of collagen and elastin with heparin in vivo6 cannot be explained at the mRNA level. It seems more likely that heparin inhibits ECM translation, post-translational processing,3435 or degradation.36 We have previously proposed that heparin might affect the turnover of ECM by a mechanism of directly inhibiting SMC proteolytic enzyme expression.37'38 In conclusion, we report inductions of ECM genes after arterial injury. These inductions occur mainly at 2 to 4 weeks after injury when the neointima gains much of its mass, although the mRNA for cell surface HSPGs begins to increase at 2 days. Our results indicate that the accumulation of ECM after injury is at least partly regulated at the mRNA level. Heparin has previously been shown to modulate ECM deposition in the neointima, but the effect does not appear to be at the level of ECM mRNA.


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Acknowledgments We thank Monika M. Clowes and Holly Henson for expert technical assistance with the rat injury model.

16.

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