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(Circulation. 1997;95:997-1006.)
© 1997 American Heart Association, Inc.

Articles

Origin of Extracellular Matrix Synthesis During Coronary Repair

Yi Shi, MD, PhD; James E. O'Brien, Jr, MD; Leena Ala-Kokko, MD, PhD; Wooksung Chung, MD; John D. Mannion, MD; Andrew Zalewski, MD

the Departments of Medicine (Cardiology), Surgery (Cardiothoracic Surgery) (J.E.O., J.D.M.), and Biochemistry (L.A.-K.), Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Dr Yi Shi, Cardiovascular Research Center, Division of Cardiology, Suite 410, Thomas Jefferson University, 1025 Walnut St, Philadelphia, PA 19107. E-mail shi1@jeflin.tju.edu.

	Abstract

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Background Coronary injury triggers differentiation of activated adventitial fibroblasts to myofibroblasts, which may contribute to neointimal formation and vascular remodeling. Accordingly, the purpose of this study was to examine the cellular origin of the enhanced synthesis of extracellular matrix proteins during coronary repair.

Methods and Results The time course and localization of collagen and elastin expression were examined by in situ hybridization and immunohistochemistry in porcine coronary arteries after balloon-induced injury. Procollagen-₁(I) transcripts and intracellular type I procollagen protein increased in the adventitia within 2 days after injury. This was followed by a sustained synthesis of type I procollagen in neointima beginning at 7 days and the extracellular accumulation of type I collagen in both layers. The origin of synthetic cells was further examined by colocalization of type I procollagen and bromodeoxyuridine labeling to activated adventitial cells, which translocated to neointima. Neointimal cells exhibited sustained synthetic activity manifested by the presence of type I procollagen and elastin at 3 months after injury. In contrast, the media showed only minor changes in the synthesis of collagen or elastin throughout coronary repair.

Conclusions Activated adventitial fibroblasts are endowed with synthetic capabilities after coronary injury. They express type I procollagen, with some of them translocating to the intima, where they continue to synthesize procollagen. The accumulation of type I collagen is evident in the adventitia and neointima, whereas elastin accumulates mainly in neointima. These findings support the involvement of adventitial fibroblasts in coronary repair and remodeling after endoluminal injury.

Key Words: angioplasty • collagen • restenosis • remodeling

	Introduction

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The accumulation of several components of the ECM is an integral part of arterial repair.^{1 2} This process reflects broader phenotypic changes of vascular cells in response to paracrine and autocrine stimuli after injury. The subsequent increase in lesion size has been associated with the elevated content of structural proteins in the vascular wall, such as collagens and elastin.^{3 4} In addition, ECM proteins affect the fate of lesions, modulating cell migration, the activity of growth factors, and local concentrations of atherogenic lipoproteins.^{5 6 7} Cell-matrix interactions may also provide the basis for constrictive remodeling, which resembles a similar phenomenon after nonvascular tissue injury.^{8 9} Recent studies from our laboratory^{10 11} and by others^{12 13} have suggested that "nonmuscle" cells contribute to arterial repair. In particular, coronary endoluminal injury invokes cell proliferation and migration involving adventitial fibroblasts. These responses are associated with their differentiation to myofibroblasts, manifested by a rapid induction of -SM actin expression.^{10 11 13} The importance of adventitial fibroblasts in arterial repair, however, has not been firmly established, because little is known regarding their synthetic properties. In general, prior studies either have analyzed ECM content in injured vessels without spatial considerations or have focused primarily on neointima.^{3 4 14} If adventitial fibroblasts are ultimately involved in arterial repair and remodeling, it is expected that the changes in synthetic activity would be associated with their activation and translocation. Hence, in the present study, we addressed the cellular origin, distribution, and time course of the increase in collagen and elastin after coronary injury. At early stages, adventitial fibroblasts exhibited augmented expression of type I collagen, which continued in the neointima. At later stages, elastin expression was increased predominantly in neointimal cells, suggesting ongoing remodeling after coronary injury. These findings reflect unique synthetic characteristics of vascular myofibroblasts derived from the adventitial fibroblasts that are involved in coronary repair and remodeling.

	Methods

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Animal Model
A porcine model of coronary arterial injury was used as described.^{10 11} Domestic crossbred pigs (21 to 33 kg) were sedated with ketamine (20 mg/kg IM) and xylazine (4 mg/kg IM). After endotracheal intubation, they were ventilated with isoflurane (1.75%) and oxygen to maintain anesthesia throughout the experiment. The coronary arteries (41 animals/115 arteries) were injured with an oversized balloon (3.5 to 4.0 mm) inflated three times (6 to 10 atm) for 30 seconds, whereas noninstrumented coronary arteries (4 animals/12 arteries) served as controls. Postsurgical therapy included aspirin 325 mg PO and ampicillin 250 mg IM for the next 2 days. To trace migration of proliferating cells in vivo, 11 pigs received BrdU (Boehringer Mannheim) at 12 hours (30 mg/kg IM) and 24 hours (30 mg/kg IV) after the procedure as described.¹¹ The animals were euthanatized with an intravenous overdose of Euthasol (Delmarva Laboratory) containing pentobarbital sodium (1950 mg) and phenytoin sodium (250 mg) at times indicated in the text. All experiments conformed to the position of the American Heart Association on research animal use and were in accordance with institutional guidelines.

Tissue Preparation
Epicardial coronary arteries were removed along with adjacent tissues and immersed in HistoChoice tissue fixative (Amresco) for >5 hours. The tissues were sectioned into 3-mm blocks, processed in a Tissue-Tek VIP processor (Miles Inc), and embedded in paraffin. The location of medial injury was determined with Verhoeff's stain in 5-µm-thick sections.¹⁵ For immunohistochemistry, sections were adhered to glass slides coated with gelatin. For in situ hybridization, specimens were adhered to charged and precleaned ProbeOn Plus slides (Fisher Scientific). The arteries from at least three animals were obtained at each time point. The number of arteries examined for each parameter is reported as an "n" value in the "Results" section.

In Situ Hybridization
Probe Preparation
The cDNA probes were prepared to detect mRNAs for types I and III procollagen-₁ chains. To avoid cross-hybridization between different collagen mRNAs, the probes were prepared so that they covered the 3' end of the corresponding mRNA sequences.^{16 17} To prepare the procollagen-₁(I) probe, an Hf677 cDNA clone (ATCC 61323) was amplified by PCR using primers COL1F (5'-GTG AGG ATC CCA GCG CTG GTT TCG ACT TC) and COL1R (5'-CAC TAA GCT TGG TCA TGT TCG GTT GGT CAA AG).¹⁸ The 1036-bp PCR product consisted of 820 bp of 3'-coding sequences and extended 198 bp beyond the codon for translation termination. A probe for procollagen-₁(III) mRNA was prepared by PCR amplification of an Hf934 cDNA clone (ATCC 61325) with the primers COL3F (5'-GTG AGG ATC CTC GAG GTA ACA GAG GTG AAA G) and COL3R (5'-TCA CAA GCT TGA TCA GGA CCA CCA ATG TC).¹⁹ The 917-bp PCR product covered the region between 906 and 35 bp before the translation termination. The 5' end of the forward primers contained a site for the restriction endonuclease BamHI, and the 5' end of the reverse primers contained a site for the restriction endonuclease HindIII. The PCR products were cloned into a plasmid vector [pGEM-7Zf(+), Promega]. The above probes were >60% sequence-specific for procollagen-₁(I) and -₁(III), respectively. The human tropoelastin cDNA clone in pT7T3D-PAC vector was used (ATCC 335298). The labeled riboprobe probes were generated by in vitro transcription. Briefly, plasmid DNA was linearized, purified by phenol-chloroform extraction, and transcribed with respective RNA polymerases by use of digoxigenin-labeled UTP as substrate (Boehringer Mannheim). The yield of transcripts was estimated by electrophoresis and ethidium bromide staining. The labeling efficiency was examined by dot blot.

Hybridization
The tissue sections were deparaffinized and immersed in 2xSSC for 10 minutes. After prehybridization for 2 hours at 42°C in solution containing 5xSSC, 0.1% N-lauroylasarcosine, 0.02% SDS, 50% formamide, and 2% blocking reagent (Boehringer Mannheim), the sections were hybridized with digoxigenin-labeled riboprobe (100 ng/mL) for 16 hours at 42°C in a MicroProbe humid chamber (Fisher Scientific). The sections were washed twice in 2xSSC for 30 minutes at 42°C and then treated with DNase-free RNase (100 µg/mL, Sigma) in 2xSSC for 30 minutes to eliminate unbound probe. After three washings in 0.1xSSC in 0.1% SDS for 20 minutes, sections were incubated with anti–digoxigenin–alkaline phosphatase conjugate (1:500) for 5 hours in a humid chamber. After the washings, sections were incubated with freshly prepared substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for 48 hours. Finally, slides were counterstained with nuclear fast red (Vector Laboratory). Negative controls included sense probe instead of antisense probe and RNase digestion of tissue for 30 minutes before hybridization with antisense probe.

Immunohistochemistry
The immunohistochemistry was carried out as described.^{10 11} The specimens were incubated with primary antibodies for 1 hour, then with biotinylated secondary horse anti-mouse antibodies (1:2000, Vector Laboratories), and the immunostaining was visualized with DAB substrate followed by counterstain with hematoxylin. Negative controls were carried out with nonimmune serum. For BrdU staining, sections were treated with 2N HCl at 37°C for 30 minutes, followed by 0.1 mol/L borax for 10 minutes before primary antibody. The following primary antibodies were used: mouse anti-sheep monoclonal antibody SP1.D8 recognizing the NH₂-terminal of type I procollagen (1:50, Developmental Studies Hybridoma Bank)²⁰ ; mouse anti-human monoclonal antibody against the COOH-terminal of type I procollagen (1:50, Chemicon International)²¹ ; monoclonal anti–collagen type I antibody (1:2000, Sigma Biosciences); monoclonal anti-elastin antibody recognizing both soluble and insoluble elastin (1:4000, Sigma Biosciences); and monoclonal mouse anti-BrdU antibody (1:200, Novocastra). For double immunohistochemistry, sections were stained first with primary antibody against BrdU as described above. After incubation with DAB substrate, they were washed and blocked with 5% horse serum. Afterward, slides were incubated with SP1.D8 antibody, which was visualized with VIP substrate.

Microscopic images at various objective magnifications were digitized with a Sony DXC-750MD video camera attached to a Nikon Optiphot-2 microscope and printed on a Sony UP5000 color video printer without additional image manipulations.

	Results

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Procollagen-₁(I) and -₁(III) by In Situ Hybridization
The uninjured coronary arteries (n=6) displayed a low level of procollagen-₁(I) transcripts in medial SM cells and in adventitial fibroblasts by in situ hybridization. At 2 to 3 days after injury, the arteries with a complete medial disruption exhibited a marked increase in procollagen-₁(I) mRNA in the adventitia and in the perivascular area adjacent to medial disruption (n=8, Fig 1). In contrast, coronary injury devoid of medial disruption (n=3) was accompanied by no significant changes regarding procollagen-₁(I) expression (not shown). At 7 to 14 days after injury resulting in medial dissection (n=6), procollagen-₁(I) transcripts decreased in the adventitia, whereas augmented procollagen-₁(I) expression was evident in the neointima (Fig 1). At 1 to 3 months after injury (n=6), procollagen-₁(I) mRNA expression returned to the baseline level in remodeled coronary arteries (not shown). In contrast to the above dynamic changes, procollagen-₁(I) transcripts in the media remained unchanged at all times. When adjacent coronary sections were probed for procollagen-₁(III) transcripts, a low-level expression was observed in the adventitia, media, and neointima for up to 3 months during the vascular repair process (Fig 2).

View larger version (90K): [in this window] [in a new window]   Figure 1. Facing page. Procollagen-1(I) mRNA expression after coronary injury. A, Control coronary artery (left, Verhoeff's stain; right, in situ hybridization) shows no significant hybridization with procollagen-1(I) antisense probe. B, At 3 days after injury (left, Verhoeff's stain; right, in situ hybridization), procollagen-1(I) mRNA is markedly elevated in the adventitia in the vicinity of medial disruption. Note no significant hybridization in the media. C, At 14 days after injury (left, Verhoeff's stain; right, in situ hybridization), the expression of procollagen-1(I) mRNA is evident in the neointima, whereas its level in the adventitia returned toward baseline. The media continues to show no significant increase in procollagen-1(I) expression. D and E, Controls for in situ hybridization. Sections adjacent to B were treated with RNase (D) or hybridized with sense probe (E) and demonstrated no hybridization with antisense probe for procollagen-1(I) mRNA. a indicates adventitia; m, media; and n, neointima. Magnification x20.

View larger version (70K): [in this window] [in a new window]   Figure 2. Procollagen-1(III) mRNA expression after coronary injury. At 3 days after injury, a low expression of procollagen-1(III) transcripts is present in the adventitia (left) and in the media (right), which resembles basal levels in controls (not shown). Arrows point to infrequent cells showing procollagen-1(III) transcripts; asterisk identifies external elastic lamina. Abbreviations as in Fig 1. Magnification x800.

Type I Collagen by Immunohistochemistry
Intracellular type I procollagen was detected by use of antibodies recognizing the NH₂-terminal (Fig 3). Infrequent adventitial fibroblasts demonstrated procollagen immunoreactivity in uninjured vessels (n=6). At 2 to 3 days after injury (n=8), a dramatic increase in type I procollagen was evident in the adventitia and perivascular area, consistent with the upregulation of procollagen-₁(I) transcripts. Likewise, type I procollagen was detected in neointimal cells beginning at 7 days (n=6). The synthesis of type I procollagen in the adventitia markedly decreased at 1 month after injury, whereas neointimal cells exhibited sustained immunoreactivity with type I procollagen antibodies for at least 3 months (n=6). In contrast, no major changes regarding procollagen I were noted in the media. Similar results were obtained with antibodies against type I procollagen COOH-terminal (not shown).

View larger version (136K): [in this window] [in a new window]   Figure 3. Procollagen-1(I) (intracellular) immunostaining after coronary injury. A, Control coronary artery shows a weak immunoreactivity with procollagen-1(I) (SP1.D8 antibody against NH2-terminal) in adventitial and medial cells. B, At 3 days after injury, activated adventitial fibroblasts exhibit a high expression of intracellular procollagen-1(I) (brown stain). C, At 8 days after injury, adventitial cells continue to demonstrate increased levels of intracellular procollagen-1(I); note that neointimal cells exhibit similar levels of procollagen-1(I). D, At 3 months after injury, a number of neointimal cells show sustained immunoreactivity with procollagen-1(I) antibody. Abbreviations as in Fig 1. Asterisk identifies external elastic lamina. Magnification x330.

Extracellular (mature) type I collagen demonstrated a marked increase in the adventitia and neointima at 3 months after injury, whereas the media showed no major changes compared with control arteries (Fig 4).

View larger version (136K): [in this window] [in a new window]   Figure 4. Type I collagen (extracellular) immunostaining after coronary injury. A, Control coronary artery shows a weak immunostaining in the adventitia and in the media. B, At 3 months after injury, remodeled coronary artery displays enhanced immunostaining in thickened adventitia and neointima. Media shows immunoreactivity similar to control. C and D are high-power photomicrographs showing the neointima (C) and the adventitia (D) identified by asterisk in B. Note intense immunostaining with type I collagen antibody in neointima and adventitia. In particular, in the adventitia, reorganized matrix has appearance of a scar. Abbreviations as in Fig 1. Magnification: A and B, x25; C and D, x210.

Migration of Synthetic Cells
The concordant changes regarding type I procollagen synthesis in the adventitia and neointima may be due to similar events in cells of different origin or may be indicative of the adventitial origin of synthetic cells in the neointima. To this end, we used BrdU labeling at 12 and 24 hours after injury to determine whether procollagen synthesis is localized to replicating fibroblasts. In addition, we attempted to identify the migration of synthetic cells to neointima. With double immunohistochemistry, a number of BrdU-labeled cells in the adventitia demonstrated positive immunoreactivity with type I procollagen antibody at 3 days after injury (n=4, Fig 5). At 7 to 14 days (n=3), these cells were present in the adventitia and neointima and colocalized type I procollagen. Their myofibroblast phenotype was confirmed by the presence of -SM actin (not shown). The media contained only few BrdU-positive cells at all times, with rare immunoreactivity with type I procollagen antibodies.

View larger version (257K): [in this window] [in a new window]   Figure 5. Colocalization of BrdU and procollagen-1(I) by double immunostaining. A, At 3 days after injury, BrdU-labeled cells (brown nuclear stain) are localized in the adventitia. The majority of BrdU-positive cells colocalize procollagen-1(I) (purple cytoplasmic stain). B, At 8 days after injury, double-stained cells are present in adventitia and neointima. Adjacent media is shown in C. C, Media demonstrates paucity of double immunostaining. Regenerating endothelial cells are positive for BrdU. Arrows point to double-stained cells. Abbreviations as in Fig 1. Asterisk identifies external elastic lamina. Magnification x1300.

Tropoelastin/Elastin by In Situ Hybridization and Immunohistochemistry
Tropoelastin transcripts were not detectable in normal coronary arteries by in situ hybridization (n=6). An increased level of tropoelastin mRNA, however, was noted in neointima at 1 to 3 months after coronary injury, whereas the media and adventitia showed no augmented hybridization (n=6, Fig 6).

View larger version (121K): [in this window] [in a new window]   Figure 6. Tropoelastin mRNA expression by in situ hybridization. An increase in tropoelastin transcripts is detected in neointima at 3 months after coronary injury (left), whereas adjacent media shows no hybridization with antisense probe (right). Arrows point to neointimal cells expressing tropoelastin mRNA. Magnification x800.

Elastin immunostaining was present mainly in the inner layer of the adventitia in normal coronary arteries (n=6, Fig 7). A paucity of elastin immunoreactivity was observed in neointima up to 14 days after injury (n=14). No major changes were noted in thickened adventitia. At 1 to 3 months after injury, an enhanced elastin immunoreactivity was evident in the neointima, whereas the media exhibited no major changes (n=6, Fig 7).

View larger version (42K): [in this window] [in a new window]   Figure 7. Elastin expression by immunostaining after coronary injury. In low-power photomicrographs (left), asterisks identify areas of interest, shown on higher magnification (right). A, Control coronary artery displays dense immunostaining in inner adventitia and weak stain in media. B, At 14 days after injury, the paucity of elastin immunoreactivity is seen in the neointima. Interrupted edges of media show a mild increase in elastin. C, At 3 months after injury, markedly enhanced elastin immunoreactivity is apparent in neointima. Staining pattern in neointima differs from that in normal media and adventitia. Abbreviations as in Fig 1. Magnification: A, x30 and x125; B, x25 and x80; C, x20 and x105.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Arterial repair involves coordinated interactions among cells, cytokines, and ECM proteins. This study shows early upregulation of type I collagen expression after coronary injury, whereas an increase in elastin expression was evident later. Importantly, the ECM synthesis was localized primarily to adventitial and neointimal cells. This spatial arrangement further implicated the adventitial origin of at least some synthetic cells during arterial repair and remodeling.

Type I collagen represents a major structural protein of the normal and diseased arterial wall.^{22 23} As shown in the present study, procollagen-₁(I) transcripts and protein were upregulated in adventitial fibroblasts after coronary endoluminal injury before the development of neointima. Although the induction of synthetic activity has been well recognized after vascular injury, such rapid increase and its location have not previously been appreciated in other models of arterial repair.^{3 4 14} Our findings, however, are consistent with the scattered observations that adventitial fibroblasts are endowed with the ability to increase type I procollagen expression in response to a variety of stimuli ranging from mechanical forces to immune injury.^{24 25 26} The sequential upregulation of collagen biosynthesis in the adventitia and neointima could be explained by the distinct events affecting two cell populations (ie, adventitial fibroblasts and dedifferentiated SM cells) in the injured vessel. Alternatively, the observed changes could reflect the contribution of adventitial cells to the repair process in the regions remote from their origin. In fact, the paucity of major changes in the adjacent media and the translocation of synthetic cells from the adventitia to neointima, as evidenced by colocalization of type I procollagen and BrdU immunostaining, suggested unique characteristics of adventitial fibroblasts during vascular repair. It is noteworthy that the accumulation of mature collagen (ie, scar) depends not only on procollagen-₁(I) gene expression but also on its postranslational processing and collagen degradation.²⁷ Despite increased intracellular procollagen-₁(I), mature type I collagen did not appear in the adventitia for 1 week after injury. This may reflect its initial extracellular degradation due to the activation of matrix metalloproteinases and plasminogen activators.^{28 29} The accumulation of mature type I collagen, however, became evident in both the adventitia and the neointima with a dense scarlike appearance, particularly in remodeled adventitia at 1 to 3 months.

We have previously shown that coronary arterial injury induces differentiation of adventitial cells to myofibroblasts.¹⁰ This response is not unique to vascular adventitia, since myofibroblast formation has been recognized in a wide range of tissue repair.^{30 31} Transforming growth factor-ß₁, the inducer of myofibroblast phenotype in nonvascular tissues,^{32 33} is not only released from platelets at the site of injury but also expressed in an autocrine manner by activated adventitial fibroblasts and then myofibroblasts.³⁴ Consistent with the profibrotic effects of this cytokine, ultrastructural characteristics of coronary myofibroblasts appear to reflect their robust synthetic activity.³⁴ In wound healing, proliferating fibroblasts migrate, acquire -SM actin, and continue to fill the gap in injured tissues by means of synthetic and remodeling properties.^{30 31 35 36} The latter may be responsible for constrictive geometric remodeling of injured vessels.^{37 38 39 40} In fact, both the adventitia and neointima express ₁ß₁-integrin after vascular injury, which may mediate myofibroblast-dependent collagen matrix reorganization.⁴¹ The presence of myofibroblasts, however, is short-lived in the adventitia,¹⁰ which is reflected by a similar time course of collagen biosynthesis. The ultimate fate of adventitial fibroblasts that migrate to the neointima remains to be determined. A sustained presence of -SM actin in neointima distinguishes arterial repair from wound healing. This may reflect a prolonged presence of myofibroblasts or dedifferentiated SM cells remaining after myofibroblast removal by apoptosis. As shown in this study, the expression of type I procollagen and elastin was evident at the time when the adventitia has become quiescent (ie, 3 months). Late accumulation of elastin was particularly interesting, because it points to the possibility of ongoing remodeling during a chronic phase of coronary repair. The persistence of synthetic activity may reflect unique signaling mechanisms due to either humoral or mechanical factors that are known to sustain myofibroblast phenotype.⁹

Contrary to the accepted paradigm, medial SM cells appear to be less responsive to injury in some experimental systems.^{10 11 12 13} Undoubtedly, the acquisition of SM markers by activated fibroblasts, their presence in the media in some vascular beds, and the possibility of species differences regarding arterial repair (eg, rat model) add to the difficulty in recognizing the role of undifferentiated "nonmuscle" cells. The increased synthesis of ECM proteins in the adventitia and neointima but not in the media lends support to our previous findings that the neointima is enriched by adventitial fibroblasts.¹¹ These lesions share some similarities with human atherosclerotic intima, mainly the presence of synthetic SM-like cells and the preponderance of type I collagen and elastin.^{1 42}

The failure of several pharmacological approaches to prevent restenosis in clinical trials can be traced to an incomplete understanding of the cellular mechanisms of arterial repair. The present study provided additional evidence that adventitial fibroblasts/myofibroblasts play an important role in coronary arterial repair.^{10 11 13 40} This is particularly relevant in light of newer transcatheter approaches, including stenting or atherectomy, which create conditions for the activation of adventitial cells. It is conceivable that the targeting of activated fibroblasts is necessary to modulate several ensuing events, such as cell proliferation, migration, and synthetic capabilities,⁴³ all associated with these unique vascular cells. The above observations may have important implications for the emerging concept of local drug delivery, because the administration of such compounds to the adventitial layer may be required to augment therapeutic effects.^{44 45}

Conclusions
Adventitial fibroblasts responded to coronary injury with a rapid induction of type I procollagen expression (mRNA and protein). After some cells translocated to the neointima, they continued to exhibit synthetic activity. The above events resulted in the deposition of mature type I collagen in both the adventitia and neointima. Late remodeling changes were also due to the accumulation of elastin in neointima. In contrast, adjacent medial SM cells demonstrated no major changes throughout vascular repair. These findings underscored the importance of adventitial fibroblasts in the process of arterial remodeling.

	Selected Abbreviations and Acronyms

 

BrdU = 5-bromo-2'-deoxyuridine ECM = extracellular matrix PCR = polymerase chain reaction SM = smooth muscle

	Acknowledgments

 
This study was supported in part by grants from the National Institutes of Health (HL-44150) and the American Heart Association, Delaware and Florida Affiliates, Inc. The authors acknowledge the excellent technical assistance of Dian Wang and Felicia Hayes. The SP1.D8 antibody was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md, and the Department of Biological Sciences, University of Iowa, Iowa City, under contract N01-HD-6-2915 from the National Institute of Child Health and Human Development.

Received July 29, 1996; revision received September 25, 1996; accepted October 5, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ross R, Wight TN, Strandness E, Thiele B. Human atherosclerosis, I: cell constitution and characteristics of advanced lesions of the superficial femoral artery. Am J Pathol. 1984;114:79-93.[Abstract]
   
2. 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]
   
3. 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. Circulation. 1995;91:2049-2057.[Abstract/Free Full Text]
   
4. Straus 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]
   
5. Knox P, Crooks S, Rimmer CS. Role of fibronectin in the migration of fibroblasts into plasma clots. J Cell Biol. 1986;102:2318-2323.[Abstract/Free Full Text]
   
6. Streuli CH, Schmidhauser C, Kobrin M, Bissell MJ, Derynck R. Extracellular matrix regulates expression of the TGF-ß1 gene. J Cell Biol. 1993;120:253-260.[Abstract/Free Full Text]
   
7. Cardoso LEM, Mourao PAS. Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL. Arterioscler Thromb. 1994;1:115-124.
   
8. Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LF, Wong SC, Hong MK, Kovach JA, Leon MB. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation. 1996;94:35-43.[Abstract/Free Full Text]
   
9. Grinnell F. Fibroblasts, myofibroblasts, and wound contraction. J Cell Biol. 1994;124:401-404.[Free Full Text]
   
10. Shi Y, Pieniek M, Fard A, O'Brien J, Mannion JD, Zalewski A. Adventitial remodeling following coronary arterial injury. Circulation. 1996;93:340-348.[Abstract/Free Full Text]
    
11. Shi Y, O'Brien JE, Fard A, Mannion JD, Wang D, Zalewski A. Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries. Circulation. 1996;94:1655-1664.[Abstract/Free Full Text]
    
12. Holifield B, Helgason T, Jemelka S, Taylor A, Navran S, Allen J, Seidel C. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest. 1996;97:814-825.[Medline] [Order article via Infotrieve]
    
13. Scott NA, Cipolla GD, Ross CE, Dunn B, Martin FH, Simonet L, Wilcox JN. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation. 1996;93:2178-2187.[Abstract/Free Full Text]
    
14. Majesky MW, Lindner V, Twardzik DR, Schwartz SM, Reidy MA. Production of transforming growth factor ß1 during repair of arterial injury. J Clin Invest. 1991;88:904-910.
    
15. McElroy DA. Connective tissue. In: Prophet EB, Mills B, Arrington JB, Sobin LH, eds. Laboratory Methods in Histotechnology. Washington, DC: American Registry of Pathology; 1992:132-135.
    
16. Dion AS, Myers JC. COOH-terminal properties of the major human procollagens: structural, functional and genetic comparisons. J Mol Biol. 1987;193:127-143.[Medline] [Order article via Infotrieve]
    
17. Metasaranta M, Toman D, De Crombrugghe B, Vuorio E. Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs. Biochem Biophys Acta. 1991;1089:241-243.[Medline] [Order article via Infotrieve]
    
18. Bernard MP, Chu M-L, Myers JC, Ramirez F, Eikenberry EF, Prockop DJ. Nucleotide sequences of complementary deoxyribonucleic acids for the pro1 chain of human type I procollagen: statistical evaluation of structures that are conserved during evolution. Biochemistry. 1983;22:5213-5223.[Medline] [Order article via Infotrieve]
    
19. Ala-Kokko L, Kontusaari S, Baldwin CT, Kuivaniemi H, Prockop DJ. Structure of cDNA clones coding for the entire prepro1(III) chain of human type III procollagen: differences in protein structure from type I procollagen and conservation of codon preferences. Biochem J. 1989;260:509-516.[Medline] [Order article via Infotrieve]
    
20. Froeilmer HG, Kawahara K, Madri JA, Furthmayr H, Timpi R, Tuderman L. A monoclonal antibody specific for the amino terminal cleavage site of procollagen type I. Eur J Biochem. 1983;134:183-189.[Medline] [Order article via Infotrieve]
    
21. McDonald JA, Broekelmann TJ, Matheke ML, Crouch E, Koo M, Kuhn C. A monoclonal antibody to the carboxyterminal domain of procollagen type I visualizes collagen-synthesizing fibroblasts: detection of an altered fibroblast phenotype in lungs of patients with pulmonary fibrosis. J Clin Invest. 1986;78:1237-1244.
    
22. Barnes MJ. Collagens in atherosclerosis. Collagen Rel Res. 1985;5:65-97.
    
23. Rekhter MD, Zhang K, Narayanan AS, Phan S, Schork MA, Gordon D. Type I collagen gene expression in human atherosclerosis. Am J Pathol. 1993;143:1634-1648.[Abstract]
    
24. Kolpakov V, Rekhter MD, Gordon D, Wang WH, Kulik TJ. Effect of mechanical forces on growth and matrix protein synthesis in in vitro pulmonary artery: analysis of the role of individual cell types. Circ Res. 1995;77:823-831.[Abstract/Free Full Text]
    
25. Rekhter MD, Yoon S, Zukauskas V, Gordon D. Type I collagen gene expression in a rabbit aortic allograft model of transplant atherosclerosis. FASEB J. 1995;9:A855. Abstract.
    
26. Wiggins R, Goyal M, Merritt S, Killen PD. Vascular adventitial cell expression of collagen I messenger ribonucleic acid in anti-glomerular basement membrane antibody-induced crescentic nephritis in the rabbit. Lab Invest. 1993;68:557-565.[Medline] [Order article via Infotrieve]
    
27. Laurent G. Dynamic state of collagen: pathways of collagen degradation in vivo and their possible role in regulation of collagen mass. Am J Physiol. 1987;252(Cell Physiol 21):C1-C9.
    
28. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994;75:539-545.[Abstract/Free Full Text]
    
29. Clowes AW, Clowes MM, Au YPT, Reidy MA, Belin D. Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery. Circ Res. 1990;67:61-67.[Abstract/Free Full Text]
    
30. Darby I, Skalli O, Gabbiani G. -Smooth muscle actin is transiently expressed in by myofibroblasts during experimental wound healing. Lab Invest. 1990;63:21-29.[Medline] [Order article via Infotrieve]
    
31. Welch MP, Odland GF, Clark RAF. Temporal relationship of F-actin bundle formation, collagen and fibronectin matrix assembly and fibronectin receptor expression to wound contraction. J Cell Biol. 1990;110:133-145.[Abstract/Free Full Text]
    
32. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G. Transforming growth factor-ß1 induces -smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol. 1993;122:103-111.[Abstract/Free Full Text]
    
33. Ronnov-Jessen L, Petersen OW. Induction of -smooth muscle actin by transforming growth factor-ß1 in quiescent human breast gland fibroblasts: implications for myofibroblast generation in breast neoplasia. Lab Invest. 1993;68:696-707.[Medline] [Order article via Infotrieve]
    
34. Shi Y, O'Brien JE Jr, Fard A, Zalewski A. Transforming growth factor ß1 expression and myofibroblast formation during arterial repair. Arterioscler Thromb Vasc Biol. 1996;16:1298-1305.[Abstract/Free Full Text]
    
35. Montesano R, Orci L. Transforming growth factor ß stimulates collagen-matrix contraction by fibroblasts: implications for wound healing. Proc Natl Acad Sci U S A. 1988;85:4894-4897.[Abstract/Free Full Text]
    
36. Arora PD, McCulloch CAG. Dependence of collagen remodelling on -smooth muscle actin expression by fibroblasts. J Cell Physiol. 1994;159:161-175.[Medline] [Order article via Infotrieve]
    
37. Post MJ, Borst C, Kuntz RE. The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty: a study in the normal rabbit and the hypocholesterolemic Yucatan micropig. Circulation. 1994;89:2816-2821.[Abstract/Free Full Text]
    
38. Kakuta T, Currier JW, Haudenschild CC, Ryan TJ, Faxon DP. Differences in compensatory vessel enlargement, not intimal formation, account for restenosis after angioplasty in the hypercholesterolemic rabbit model. Circulation. 1994;89:2809-2815.[Abstract/Free Full Text]
    
39. Lafont A, Guzman LA, Whitlow PL, Goormastic M, Cornhill JF, Chisolm GM. Restenosis after experimental angioplasty: intimal, medial and adventitial changes associated with constrictive remodeling. Circ Res. 1995;76:996-1001.[Abstract/Free Full Text]
    
40. Andersen HR, Mæng M, Thorwest M, Falk E. Remodeling rather than neointimal formation explains luminal narrowing after deep vessel wall injury. Circulation. 1996;93:1716-1724.[Abstract/Free Full Text]
    
41. Gotwals PJ, Chi-Rosso G, Lindner V, Yang J, Ling L, Falwell SE, Koteliansky VE. The 1ß1 integrin is expressed during neointima formation in rat arteries and mediates collagen matrix reorganization. J Clin Invest. 1996;97:2469-2477.[Medline] [Order article via Infotrieve]
    
42. Botney MD, Kaiser LR, Cooper JD, Mecham RP, Parghi D, Roby J, Park WC. Extracellular matrix protein gene expression in atherosclerotic hypertensive pulmonary arteries. Am J Pathol. 1992;140:357-364.[Abstract]
    
43. Shi Y, Fard A, Galeo A, Hutchinson H, Vermani P, Dodge GR, Hall DJ, Sheehan F, Zalewski A. Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary balloon injury. Circulation. 1994;90:944-951.[Abstract/Free Full Text]
    
44. Rogers C, Karnovsky MJ, Edelman ER. Inhibition of experimental neointimal hyperplasia and thrombosis depends on the type of vascular injury and the site of drug administration. Circulation. 1993;88:1215-1221.[Abstract/Free Full Text]
    
45. Nathan A, Nugent MA, Edelman ER. Tissue engineered perivascular endothelial cell implants regulate vascular injury. Proc Natl Acad Sci U S A. 1995;92:8130-8134.[Abstract/Free Full Text]
    

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