EXPERIMENTAL STUDY
Adventitial remodeling after angioplasty is associated with expression of tenascin mRNA by adventitial myofibroblasts
Kurt Wallner, MD*,
Behrooz G. Sharifi, PhD*,
Prediman K. Shah, MD, FACC*,
Sumiko Noguchi, MD ,
Hector DeLeon, MD, PhD and
Josiah N. Wilcox, PhD
* Atherosclerosis Research Center, Division of Cardiology, Burns and Allen Research Institute, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California,, USA
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA
Manuscript received May 18, 2000;
revised manuscript received August 29, 2000,
accepted October 4, 2000.
Reprint requests and correspondence: Dr. Behrooz G. Sharifi, Cedars-Sinai Medical Center, Davis Building #1016, 8700 Beverly Boulevard, Los Angeles, California 90048
Sharifi{at}CSMC.edu
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Abstract
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OBJECTIVES
The purpose of this study was to determine the temporospatial expression of tenascin-C (TnC) in balloon-injured rat and porcine arteries.
BACKGROUND
Recent studies suggest that cell migration, in addition to cell proliferation, is a critical component of neointima formation after vascular injury. We have previously shown that adventitial myofibroblasts synthesize growth factors that contribute to the formation of neointima after arterial injury. We have also shown that the extracellular matrix protein, TnC, regulates cell migration. Consequently, we investigated the temporospatial expression of TnC by myofibroblasts after vascular injury.
METHODS
In situ hybridization and immunohistochemistry were used to investigate the temporospatial expression of TnC in injured arteries. Northern and Western blots were used to determine the in vitro expression of TnC.
RESULTS
In situ hybridization revealed that the major site of TnC expression early after vascular injury was the adventitial myofibroblasts. Immunohistochemical staining demonstrated that TnC expression began in adventitial myofibroblasts three days after injury. Tenascin-C expression, however, did not persist in this region. Rather, it moved progressively across the vascular wall toward the luminal surface. By one week, TnC expression reached the developing neointima. In vitro, myofibroblasts did not express TnC mRNA under basal conditions. In contrast, angiotensin II and PDGF-BB, factors that have been implicated in remodeling of balloon-injured arteries, markedly upregulated TnC mRNA.
CONCLUSIONS
Tenascin-C is expressed in response to balloon injury. Tenascin-C expression begins with adventitial myofibroblasts. Over a period of 7 to 14 days, expression moves progressively across the vessel wall to the neointima. We hypothesize that adventitial myofibroblasts are actively involved in the formation of neointima and that TnC facilitates migration of these cells during adventitial remodeling.
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Abbreviations and Acronyms
| | RCA | = right coronary artery | | SMC | = smooth muscle cell | | TnC | = tenascin-C |
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Proliferation and migration of medial smooth muscle cells (SMC) play a fundamental role in the formation of neointima (1). Injury to the blood vessel causes development of neointima, with significant remodeling of the adventitia, accompanied by the differentiation of adventitial fibroblasts to myofibroblasts (2–5). Conversely, localized injury to the adventitia causes neointimal formation (6).
If adventitial fibroblasts are involved in intimal hyperplasia, the presence and timing of upregulation of genes associated with cell migration should be detectable. Tenascin-C (TnC) is one of the extracellular matrix proteins that have been implicated in cell migration during embryogenesis. It is expressed in a rigidly controlled pattern of temporospatial expression in a developing fetus, yet it is typically undetectable in the corresponding regions of the intact adult organ. Increased expression of TnC has been found to be associated with the progression of clinical and experimental pulmonary hypertension (7). We have found increased expression of TnC in human atheroma (8) and arterialized human vein grafts (9). We have shown that TnC blocks adhesion of SMC to fibronectin (10) and promotes migration of arterial SMCs (11).
Previous studies suggested that medial SMCs are the source of TnC in balloon-injured rat carotid arteries (12,13). In this study, however, we demonstrate that adventitial myofibroblasts are the initial source of TnC mRNA in injured rat and porcine arteries, suggesting that these cells have a primary role in the response to vascular injury.
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Methods
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Arterial injury, rats.
Male Sprague-Dawley rats (450 to 500 g) were anesthetized, and acute injury to the left common carotid artery was made with an 2F embolectomy catheter as we previously described (14). At the indicated times after injury (6 rats/time point), animals were put to death, and both injured (left) and uninjured (right) common carotid arteries were excised and prepared for histological examination by in situ hybridization and immunohistochemistry. Arteries were prepared for histological analysis by perfusing the animals with saline and 4% paraformaldehyde at 100 mm Hg for 5 min. The vessels were dissected, dehydrated in sequential alcohols and embedded in paraffin blocks for subsequent immunostaining and in situ hybridization studies.
Arterial injury, pigs.
The model of overstretch injury has been described previously (2). Female domestic pigs (Sus scrofa, 18 to 27 kg) were given aspirin (325 mg) one day before and on the day of the procedure. Balloon overstretch injury was performed with a 3.5 mm clinical angioplasty balloon positioned in the proximal segments of the left anterior descending and left circumflex arteries with a balloon–artery ratio of 1.3 and inflated to 10 atm three times for 30 s in each artery. Inflation periods were separated by 1-min deflation periods to restore coronary perfusion. After the completion of the third inflation, the angioplasty balloon was withdrawn, and additional nitroglycerin (200 µg) was administered to limit coronary spasm. Repeat angiography was then performed to assess vessel patency and degree of injury. After injury the guide catheter was removed and the femoral cutdown repaired.
The animals were put to death with an overdose of barbiturate either 3, 7 or 14 days after injury; the heart was removed, and the injured arteries were perfused in situ with saline followed by 4% paraformaldehyde in NaPO4 buffer (pH 7.4) at 100 to 110 mm Hg pressure for 5 min. The arteries were then dissected from the heart and immersed overnight in 15% sucrose-PBS. The following day the vessels were divided into serial 3.0 mm segments and frozen in liquid nitrogen embedded in optimal cutting temperature compound (O.C.T., Miles Laboratories) in a manner allowing the serial reconstruction of each vessel. Histologic analysis was performed on 6 µm cryosections collected onto glass slides (Fisher SuperFrost Plus). The pig and rat studies were conducted with the approval of the Emory University Institutional Animal Care and Use Committee and were in accordance with all federal guidelines.
In situ hybridization.
Full-length riboprobes were transcribed as described using 35S-UTP (Amersham, specific activity >1,200 Ci/mmol), and in situ hybridization was performed as previously described (15). Briefly, cryosections were pretreated with 4% paraformaldehyde, 100 µg/ml proteinase K (Sigma) and prehybridized in 100 µl hybridization buffer (50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris pH 8.0, 5 mmol/L EDTA, 0.02% polyvinylpyrrolidone, 0.02% ficoll, 0.02% bovine serum albumin, 10% dextran sulfate and 10 mmol/L dithiothreitol) at 42°C. The hybridizations were started by adding 600,000 CPMs of 35S-riboprobe in a small volume of hybridization buffer into the prehybridization solution. The sections were then incubated at 55°C overnight. After hybridization, the sections were washed with 2 x SSC (1 x SSC = 150 mmol/L NaCl, 15 mmol/L Na citrate, pH 7.0) with 10 mmol/L beta-mercaptoethanol and 1 mmol/L EDTA, treated with 20 µg/ml heat-treated RNase A (Sigma), again washed in the same buffer and followed by a high stringency wash in 0.1 x SSC with 10 mmol/L beta-mercaptoethanol and 1 mmol/L EDTA at 55°C. The slides were then washed in 0.5 x SSC without beta-mercaptoethanol and 1 mmol/L EDTA and dehydrated in graded alcohols containing 0.3 mol/L NH4Ac. The sections were dried, coated with NTB2 nuclear track emulsion (Kodak, Rochester, New York) and exposed in the dark at 4°C for four weeks. After development, the sections were counterstained with hematoxylin and eosin to aid in cell identification and viewed on a Leitz Aristoplan photomicroscope equipped with polarized light epiluminescence to visualize the silver grains.
Immunohistochemistry.
Immunohistochemistry was performed essentially as described (8). Briefly, segments from normal and ballooned-injured rat carotid arteries (6 rats/time point) were embedded in O.C.T. compound (Tissue Tek, Miles, Inc., Elkhart, Indiana), and frozen sections were mounted onto glass slides. Preincubation was performed in a humidified chamber at room temperature with 2% ovalbumin in PBS containing 0.05% sodium azide. Primary antibody incubation was performed for 18 h at 4°C using a 1:500 dilution of anti-TnC antiserum in the preincubation buffer. After washing in PBS, the sections were incubated with biotin-conjugated goat-antirabbit IgG (Pierce, Rockford, Illinois) at room temperature for 30 min. This was followed by another PBS wash and an incubation with 2.5 mg/ml streptavidin-congugated Texas red (Pierce, Rockford, Illinois) in PBS. After 30-min incubation in a dark chamber, the sections were washed with PBS, coverslipped and photographed using a rhodamine filter and ektachrome 160T slide film (Eastman Kodak, New Haven, Connecticut).
Cell culture.
Porcine coronary artery smooth muscle and adventitial cells were isolated by the explant method. The right coronary artery (RCA) from a normal juvenile female domestic Yorkshire swine was dissected from the myocardium with its surrounding adventitial tissue and placed in sterile DMEM. The artery was then dissected free of the surrounding adventitial tissue and opened longitudinally with fine scissors in a tissue culture hood. The luminal surface was rubbed gently with a sterile cotton swab and washed with DMEM to remove the endothelial cells. To isolate a relatively pure smooth muscle layer, the inner medial surface was peeled away from the external elastic lamina using fine forceps. The adventitia layer was isolated from the fluffy white adipose tissue surrounding the RCA. The inner medial portion and the adventitial tissue were minced into small pieces (approximately 1–2 mm), and these were placed in separate culture dishes under coverslips with 1 to 2 ml of DMEM containing 10% fetal bovine serum and incubated for several days at 37°C with 5% CO2. Morphologically distinct cultures were obtained within two weeks; the adventitial cultures appeared more cobblestone-like in appearance and were contact inhibited, whereas the smooth muscle cultures grew in a typical hill and valley pattern. Cells from both sets of cultures stained with antibodies against alpha smooth muscle actin antibodies by passage three suggested that the adventitial cells were taking on a myofibroblast phenotype by this time. Cells were used between passage 5 and 10 for the present studies.
Northern and Western blot analysis.
Total RNA was extracted from cultured adventitial cells treated with angiotensin II and PDGF-BB. Northern blot was performed essentially as described before using TnC cDNA probe corresponding to the full-length fibrinogen-like domain of TnC (11). This probe was also used for the in situ hybridization studies. The probe hybridizes to both the long and short isoforms of TnC. Western blot was performed on the cultured media collected from adventitial cells treated with angiotensin II and PDGF-BB essentially as described (10) using chicken antibodies that we raised against the recombinant fibrinogen-like domain of TnC. The antibody was used at the 1:5,000 dilution.
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Results
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We used immunohistochemical analysis to explore the time-course and spatial distribution of TnC protein in the injured rat carotid arteries. Uninjured vessels were uniformly negative for TnC (Fig. 1A). Immunoreactivity remained minimal at early times (24 h) after injury (Fig. 1B). Two days after injury, however, cells located in the medial/adventitial interface preferentially stained for TnC (Fig. 1C), and a gradient of staining from this interface to the lumen became detectable at day 3 (Fig. 1D). The stain was uniformly distributed throughout the medial layer at day 5 (Fig. 1E). A gradient of staining from the media to the lumen began to develop at day 6 (not shown), followed by concentration of the stain around the lumen at day 7 (Fig. 1F). These data indicate that TnC is initially expressed in the inner layer of the artery and gradually moves toward the lumen. In addition, the data show that medial cells do not uniformly express TnC.
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Figure 1 Immunohistochemical localization of TnC in rat carotid arteries. Frozen sections of uninjured (A) as well as arteries collected two days (B), three days (C), four days (D), five days (E) and seven days (F) after injury were stained with a polyclonal anti-TnC antibody (1:500). Adjacent sections were stained with control preimmune antibodies. The lumen is toward the top of each panel. All photomicrographs are at a x100 magnification. Substitution of anti-TnC antibodies with an equivalent concentration of preimmune rabbit serum (1:500) or an irrelevant antibody produced no staining of normal or injured carotid artery sections. adv = adventitia; m = media; TnC = tenascin-C.
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Results obtained by in situ hybridization after balloon injury of the rat vessels largely confirms the localization of TnC by immunohistochemistry (Fig. 2). Tenascin-C mRNA was not detected in noninjured arteries (not shown) by 3 and 5 days after injury; however, a large number of TnC positive cells were found in the adventitia clustered around the injury site (Fig. 2A). The localization of TnC mRNA containing cells in the adventitia at this time probably accounts for the TnC protein detected in the injured vessels by immunostaining. In situ hybridization indicated that these cells were closely aligned against the external elastic lamina. In situ hybridization further indicated that 14 days after injury TnC mRNA was no longer found in the adventitia but was detected in the neointima along the luminal surface (Fig. 2B).
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Figure 2 In situ expression of TnC in injured rat arteries. TnC mRNA was detected by in situ hybridization 3 (A) or 14 (B) days after balloon injury of rat carotid artery using full-length TnC riboprobes. Cryosections were treated with proteinase K. After hybridization, the sections were washed, dried, coated and exposed in the dark at 4°C for four weeks. After development, the sections were counterstained with hematoxylin and eosin to aid in cell identification and viewed on a photomicroscope. TnC = tenascin-C.
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Balloon overstretch injury was performed on porcine coronary arteries, and the distribution of TnC protein and mRNA was determined by immunohistochemistry and in situ hybridization (Fig. 3), respectively. Normal porcine coronary arteries did not contain any TnC protein or mRNA (not shown). Three days after injury, however, there was an increase in TnC immunostaining along the luminal surface of the injured artery and in adventitial myofibroblasts surrounding the injury site (Fig. 3B). Tenascin-C mRNA was found in the adventitial myofibroblasts colocalized with TnC protein (Fig. 3, A and B). Tenascin-C mRNA, however, was not in the luminal cells or the media at this time. Seven days after balloon injury, TnC protein immunostaining was localized throughout the adventitia and neointima at the injury site, and by 14 days after injury, TnC mRNA and protein was confined to a single layer of intimal cells (Fig. 3, C and D).
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Figure 3 Localization of tenascin-C by in situ hybridization (A and C) and immunohistochemistry (B and D) after balloon overstretch injury of porcine coronary arteries. Balloon injury was performed in porcine left anterior descending coronary arteries as described in the Methods section and injured vessels harvested 3 (A and B) or 14 days (C and D) after injury. adv = adventitia; m = media.
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Our in vivo data demonstrate that adventitial cells express TnC mRNA and protein. To determine whether these cells are capable of expressing TnC in vitro, we examined the ability of adventitial cells to synthesize and secrete TnC when they are treated with factors that are implicated in the response to arterial injury. Confluent cultures of adventitial cells were treated with angiotensin II and PDGF-BB, and gene expression was analyzed by Northern blot. Tenascin-C mRNA was not detected under control conditions (1% serum). Addition of angiotensin II and PDGF-BB markedly increased the expression of two major TnC transcripts, 7.0 kb and 8.4 kb. The induction of TnC by PDGF-BB was markedly reduced at 24 h of treatment, whereas it was strongly detectable at 24 h of treatment with angiotensin II (Fig. 4A).
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Figure 4 Effect of angiotensin II and PDGF-BB on TnC mRNA and protein expression. Quiescent culture of porcine adventitial cells were treated with 1 nM PDGF-BB or 100 µM angiotensin II for the indicated times (in hours). Total RNA was extracted and analyzed by Northern blot and hybridized with TnC. To control for loading, the blot was rehybridized with the GAPDH cDNA probe. (B) For Western blot analysis, the media from cultured cells treated with angiotensin II or PDGF-BB for 8 and 24 h were collected and analyzed using 1:5,000 dilution of chicken antibodies to the recombinant fibrinogen-like domain of TnC. GAPDH = glyceraldehyde-phosphate dehydrogenase; TnC = tenascin-C.
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To determine whether the translation of the message and secretion of the protein follows the induction of TnC mRNA, the culture media were collected, and the presence of TnC was measured by Western blot. We found that adventitial cells secrete the 220 kDa and 280 kDa protein, corresponding to the small and large isoform of TnC, respectively (Fig. 4B). These data indicate that adventitial cells respond to angiotensin II and PDGF-BB treatment and secrete the small and large isoforms of TnC.
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Discussion
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Major findings.
The most important finding of this study is that early upregulation of TnC expression after coronary injury is localized primarily to adventitial cells. Tenascin-C expression began at the adventitia/medial interface two to three days after injury. Over a seven day period, the expression moved progressively from the adventitia to the media and then to intima. This temporospatial expression of TnC by myofibroblasts was similar in rats and porcine, suggesting that this event is a conserved phenomenon. The spatial expression of TnC in these animal models is also consistent with that observed in human arterial vein grafts (9), further supporting the involvement of adventitial cells in the vascular remodeling of injured arteries. The initial increased synthesis of TnC in the adventitia, which precedes neointimal formation, lends support to our previous findings that the neointima is enriched by adventitial fibroblasts (2), and the conserved adventitial expression pattern through species suggests that this is an important event in arterial response to injury.
Using immunostaining, Hedin et al. (12) found expression of TnC in the neointima of injured rat carotid artery two weeks after balloon injury. They proposed that this expression is secondary to the phenotypic modulation of neointimal SMCs. Our data, however, clearly show that TnC is initially and primarily expressed by the adventitial myofibroblasts, not by medial SMCs. Our finding is consistent with our previous observation that adventitial myofibroblasts proliferate three days after balloon injury in the porcine coronary artery. The proliferating cells are found primarily in neointima seven days after injury (2). We also have shown that myofibroblasts, which proliferate in the adventitia early after balloon injury of the coronary artery, migrate into the neointima by 14 days after angioplasty (2). We hypothesize that TnC promotes formation of neointima by facilitating migration of adventitial myofibroblasts.
Our cell culture studies establish that adventitial cells are capable of expressing TnC mRNA and protein. These cells express little TnC under basal conditions. Treatment of the cells with factors that are implicated in the repair of injured arteries, however, markedly induces TnC gene expression. This pattern of TnC expression by cultured myofibroblasts is very similar to cultured SMCs derived from the medial layer of rat aorta as reported by us and others (10,16,17). In addition, the induction of TnC mRNA by PDGF-BB and angiotensin II has kinetics similar to those we observed in the SMC culture studies; that is, the induction by PDGF-BB was transient, whereas angiotensin II had a long lasting effect. Further, the expression of individual TnC isoforms by the adventitial cells is also similar to SMCs. Both cell types expressed the two TnC mRNA transcripts corresponding to the small (7.0 kb) and large (8.4 kb) TnC isoforms.
Although it is customary to assume that medial cells are composed of SMCs, the nature of these cells remains unclear. A number of phenotypes have been described, and these phenotypes can be maintained in vitro over many passages (18–21), demonstrating the heterogeneity of the medial cells. For example, Holifield et al. (22) isolated and cultured at least two types of cells from canine carotid artery: type 1 cells, which exhibited stable expression of SMC-specific markers and did not proliferate and type 2 cells, which increased their expression of alpha-smooth muscle actin with time in culture and did proliferate. Because of the proliferative activity of type 2 cells, it was suggested that these cells are responsible for neointimal formation. Therefore, it is difficult to accurately predict the nature of the medial cell type that expresses TnC in vivo; however, the adventitial cells are clearly the initial source of TnC after balloon injury.
Summary and potential clinical implications.
Adventitial cells express TnC immediately after balloon injury. The expression has a specific and reproducible temporospatial sequence, moving progressively from adventitia to neointima over a 14-day period during the formation of neointima. These in vivo results are paralleled by the ability of cultured myofibroblasts to express the TnC gene. The data suggest that adventitial cells actively participate in the remodeling of injured vascular tissues. Since we have previously shown that TnC promotes cell migration by reducing their interaction with their extracellular matrix (11), we suggest that expression of TnC by adventitial myofibroblasts may provide a key signal for migration and proliferation of these cells and formation of neointima. The failure of several pharmacological approaches to prevent restenosis in clinical trials may be related, in part, to an incomplete understanding of the role of the adventitia in cellular mechanisms of arterial repair. We propose that therapeutic strategies to control cell growth and migration after balloon angioplasty should include consideration of the response of the adventitial cells.
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Footnotes
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Drs. Sharifi and Wilcox were supported by funding from the National Institute of Health, grants HL50566 and HL57908, respectively. Dr. Sharifi was also the recipient of an Established Investigator Award 0040201N from the American Heart Association, and Dr. Wilcox was also funded by the Georgia affiliate of the American Heart Association.
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Abstract
Methods