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Circulation. 1996;94:1655-1664

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(Circulation. 1996;94:1655-1664.)
© 1996 American Heart Association, Inc.

Articles

Adventitial Myofibroblasts Contribute to Neointimal Formation in Injured Porcine Coronary Arteries

Yi Shi, MD, PhD; James E. O'Brien, Jr, MD; Ali Fard, MD; John D. Mannion, MD; Dian Wang, BS; Andrew Zalewski, MD

the Departments of Medicine (Cardiology) and Surgery (Cardiothoracic Surgery) (J.E.O'B., J.D.M.), Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Andrew Zalewski, MD, Cardiovascular Research Center, Division of Cardiology, Thomas Jefferson University, Suite 410N, 1025 Walnut St, Philadelphia, PA 19107.


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
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Background The adventitia undergoes remodeling changes after a deep medial coronary injury. Because this process is associated with the formation of adventitial myofibroblasts, which resemble medial smooth muscle (SM) cells, we have examined myofibroblast involvement in the development of neointima.

Methods and Results In a porcine model, severe endoluminal coronary injury resulted in fibroblast proliferation and adventitial remodeling. Significant adventitial responses were associated with increased neointimal formation (P<.01). To examine the contribution of adventitial cells to the development of neointima, proliferating cells were labeled with bromodeoxyuridine (BrdU) at 12 and 24 hours after injury, and their subsequent localization was determined by immunohistochemistry (n=24). At 2 to 3 days after severe injury, the adventitia contained numerous BrdU-labeled cells (37±4%), whereas the media demonstrated infrequent labeled cells (4±1%). Adventitial cells lacked {alpha}-SM actin and desmin, which distinguished them from medial SM cells. At 7 to 8 days, some labeled cells acquired characteristics of myofibroblasts expressing {alpha}-SM actin. They were found to translocate to the gap between dissected media and contributed to the formation of neointima (76±19%). At 18 to 35 days, labeled cells were abundant in the neointima (86±5%). They showed uniform immunostaining for {alpha}-SM actin but not for desmin, thereby differing from medial SM cells and blood-borne cells.

Conclusions This study demonstrates translocation of adventitial fibroblasts to neointima, their phenotypic modulation to myofibroblasts, and distinct characteristics of myofibroblasts within neointima after severe endoluminal coronary injury. These findings suggest the significance of vascular fibroblasts in the process of arterial repair.


Key Words: adventitia • myofibroblasts • remodeling • restenosis


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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The formation of neointima represents a major mechanism of vascular repair.1 Although this process differs in various pathological conditions, proliferation and migration of medial SM cells are thought to play a fundamental role in early stages of intimal thickening.2 3 In particular, numerous experimental studies after acute medial injury have repeatedly identified SM-like cells in different vascular beds during neointimal formation.4 5 6 The development of neointima also has been observed after several adventitial manipulations, which raises the possibility of alternative mechanisms of vascular repair.7 8 9 10 Our recent findings have shown that endoluminal coronary injury results in a significant remodeling of the adventitia, accompanied by the differentiation of adventitial fibroblasts to myofibroblasts, which acquire {alpha}-SM actin.11 The presence of myofibroblasts has been a well-recognized hallmark of tissue contraction in various pathological conditions,12 13 14 and it may represent a putative mechanism of unfavorable geometric remodeling manifested by vascular constriction after balloon angioplasty.15 16 17 18

Striking similarities between myofibroblasts and SM cells in regard to their ultrastructural characteristics and the expression of cytoskeletal protein markers (eg, {alpha}-SM actin) have made distinction between these cell lines particularly difficult.19 These observations have raised the possibility that vascular myofibroblasts not only contribute to the changes affecting the adventitia11 but also may migrate toward the lumen, contributing to neointimal formation. In this study, we have demonstrated migration of adventitial cells to neointima and confirmed their transition to myofibroblasts in porcine coronary arteries after balloon injury. Myofibroblasts in neointima have shown distinct characteristics in regard to the expression of cytoskeletal proteins compared with those cells that remained in the adventitia. These findings demonstrate a new mechanism of vascular repair after a deep medial injury.


*    Methods
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 up arrowIntroduction
 *Methods
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Animal Model
Domestic crossbred pigs (Sus scrofa) weighing 23 to 31 kg were premedicated with aspirin (650 mg PO), atropine (1 mg IM), and nifedipine (10 mg sublingual). The pigs were sedated with intramuscular injection of ketamine (20 mg/kg) and xylazine (4 mg/kg). After endotracheal intubation, they were ventilated with isoflurane (1.75%) and oxygen to maintain anesthesia throughout the experiment. The right common carotid artery was surgically exposed, and heparin (10 000 U) was administered intra-arterially. The coronary ostia were cannulated under fluoroscopic guidance with an 8F SAL 1 guiding catheter, and nitroglycerin was administered (100 µg IC). The coronary arteries were injured with an oversized balloon (3.5 to 4.0 mm) inflated three times (6 to 10 atm) for 30 seconds (n=36), whereas noninstrumented coronary arteries served as controls (n=5). The site of balloon inflation was selected according to luminal dimensions of target coronary arteries that corresponded to their comparable microscopic characteristics (muscular arteries). Postsurgical therapy included aspirin 325 mg PO and ampicillin 250 mg IM for the next 2 days. The pigs were euthanatized with 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 Processing and Vascular Injury Scoring
To trace migration of proliferating cells, some animals received BrdU (Boehringer Mannheim) at 12 (30 mg/kg IM) and 24 hours (30 mg/kg IV) after the procedure. They were killed at various time points, ranging from 2 to 35 days after balloon injury, as indicated in the text. The entire epicardial porcine coronary arteries and adjacent tissues were removed in a block, rinsed with PBS, and then immersed in HistoChoice tissue fixative (Amresco) for >5 hours. The tissues were sectioned into {approx}3-mm blocks and processed in a Tissue-Tek VIP processor (Miles Inc). Then they were embedded in paraffin and cut into 5-µm-thick sections. Sections were adhered to glass slides previously coated with Vectabond (Vector Laboratories).

To determine the location and the extent of medial injury, the tissue sections were deparaffinized, and Verhoeff's stain for elastic tissues20 was used in the representative slides from each block. Severe injury was defined as the disruption of the media resulting in the exposure of the adventitia to the lumen. Mild injury was defined as punctated breaks in the IEL and/or incomplete medial damage without direct exposure of the adventitia to the lumen (Fig 1Down). Three independent observers graded the severity of arterial injury. The number of vessels examined for each parameter is reported as the n value in the "Results" section.



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  		Figure 1. Mild and severe injury after balloon overstretch in porcine coronary arteries. A, Mild injury (2 days): breaks in the IEL (arrows) without medial dissection. B, Severe injury (2 days): medial disruption results in the exposure of the adventitia to the vessel lumen. Note early adventitial thickening. C, Vascular repair after mild injury (28 days): a well-preserved continuity of the media is associated with a thin layer of neointima. D, Vascular repair after severe injury (28 days): a large neointimal lesion that fills the gap in the disrupted media is present (arrows). The adventitia demonstrates marked remodeling. a indicates adventitia; n, neointima; and t, thrombus. Verhoeff's stain. Magnification x58 (A through C) and x40 (D).

Immunohistochemistry
The Vectastain Elite ABC system (Vector Laboratories) was used for immunohistochemistry. At least 10 sections from each coronary artery with defined degree of injury (mild or severe) were analyzed. Sections were deparaffinized, incubated with 0.6% hydrogen peroxide in methanol for 30 minutes, and blocked with 5% horse serum when mouse monoclonal antibody was used. For BrdU staining, sections were treated with 2 N HCl at 37°C for 30 minutes after blocking and then incubated with 0.1 mol/L Borax (Sigma Diagnostics) for 10 minutes. After being washed in PBS, sections were incubated with primary antibodies for 1 hour at room temperature in a moisture chamber. The following primary antibodies were used: monoclonal mouse 1A4 antibody recognizing {alpha}-SM actin (1:100, Sigma Diagnostics), monoclonal mouse DE-R-11 antibody recognizing intermediate filament desmin (1:50, Novocastra), and monoclonal mouse antibody recognizing BrdU (1:200, Novocastra). Afterward, slides were washed and incubated with biotinylated secondary horse anti-mouse antibodies (1:2000, Vector Laboratories) for 1 hour. They were visualized with DAB substrate (Vector Laboratories) followed by counterstain with Gill's hematoxylin (Sigma Diagnostics). Negative controls were carried out with nonimmune serum instead of primary antibody.

For double immunohistochemistry, sections were stained first with primary antibodies against BrdU as described above. After incubation with DAB substrate, they were washed in PBS three times and blocked with 5% horse serum. Afterward, slides were incubated with the second primary antibodies (against {alpha}-SM actin or desmin) that were visualized with VIP substrate (Vector Laboratories).

Quantitative Measurements
From each coronary artery, at least three sections from different tissue blocks ({approx}3 to 5 mm apart) demonstrating only severe or mild injury were subjected to quantitative analyses. The percentage of BrdU-positive cells (dark-brown nuclear stain) was calculated in the vicinity of injury and on the opposite side for the adventitia and media. Likewise, BrdU-labeled cells were counted in neointima. In each compartment, >=500 cells were counted by use of the same objective magnification, and then mean values for multiple sections were calculated to minimize selection bias. In sections stained with Verhoeff's, neointimal and adventitial areas were measured with computerized planimetry. In vessels with severe injury, neointimal areas were delineated by the inner edge of the EEL, the border of dissected media, and the luminal edge of the neointima. In vessels with mild injury, neointimal areas were traced between the IEL, media, and luminal edge of the neointima. Adventitial areas were defined between the inner border of the EEL outward to the edge of the adipose tissue or myocardium. Repeated measurements yielded an intraobserver variability of <10%.

Statistical Analysis
All numerical data are presented as mean±SEM. One-way ANOVA was used to compare the multigroup variables. If the F test results were significant, Bonferroni's analysis was carried out to determine differences among subgroups. A value of P<.05 was required to reject the null hypothesis.


*    Results
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 *Results
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Adventitial Injury and Vascular Repair
To assess the vascular response to injury, proliferating cells within the adventitia, morphometric changes affecting the outer layer of the vessel, and the ensuing neointimal formation were quantified in porcine coronary arteries. As expected, a comparable balloon overstretch resulted in variable vascular damage (Fig 1Up). Accordingly, the comparisons were carried out between the groups with severe injury (ie, medial disruption exposing the adventitia to the lumen) and mild injury (ie, medial injury resulting in no direct contact of the adventitia with the lumen). As the TableDown shows, severe injury (n=23) was associated with significant cell proliferation within the adventitia (at 2 to 3 days) and adventitial remodeling (at 7 to 8 days), followed by increased neointimal formation (at 18 to 35 days). In contrast, mild injury (n=13) produced markedly attenuated adventitial changes and significantly smaller neointima (Fig 1Up). The above relationship between adventitial changes and the ensuing neointimal formation raised the possibility of the contribution of adventitial cells in vascular repair of a severely injured artery.


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  		Table 1. Effects of Vascular Injury on the Activation of Adventitial Cell Proliferation (2 to 3 Days), Adventitial Remodeling (7 to 8 Days), and Neointimal Formation (18 to 35 days) in Porcine Coronary Arteries

Translocation of Adventitial Cells
To examine whether translocation of adventitial cells contributes to the development of neointima after severe coronary arterial injury, BrdU was administered at 12 and 24 hours after the procedure, and the subsequent localization of labeled cells was analyzed. At 2 days after severe injury (n=6), medial dissection at the site of injury and a variable amount of thrombus constituted typical microscopic features (Fig 2ADown). As Fig 2B through 2DDown shows, most BrdU-labeled cells were circumferentially distributed in the adventitia (37±4% of adventitial cells), which contrasted with their small number in the media (4±1% of medial cells, P<.001).



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  		Figure 2. Porcine coronary arteries showing localization of BrdU-labeled cells 2 days after balloon injury. A, Overview of a severely injured coronary artery (Verhoeff's stain). Medial dissection resulting in the exposure of the adventitia to the vessel lumen is evident. A thin layer of thrombus covers the exposed EEL; no neointima has developed. B through D, BrdU immunostaining in the section adjacent to that shown in A. B, At the edge of medial disruption, single BrdU-positive cells can be seen in the media. Positive BrdU immunostaining (brown nuclear stain) is present in the adventitia. C, At the center of injury, numerous BrdU-positive cells are localized in the adventitia. D, Opposite to medial disruption, most labeled cells are present in the adventitia, with some infiltrating the EEL. Note the paucity of BrdU-positive cells in the media. Arrows point to the EEL. a indicates adventitia; m, media. Magnification x20 (A) and x200 (B through D).

At 7 to 8 days (n=4), focal adventitial thickening and a thin layer of the neointima interdigitated with residual thrombus were seen at the site of medial injury (Fig 3ADown). As Fig 3B through 3DDown shows, the cells labeled with BrdU during the first 24 hours accumulated on both sides of the EEL, with some demonstrating distorted morphology while migrating through the EEL. Numerous neointimal cells contained the label (76±19%). At 18 to 35 days (n=5), persistent thickening of the adventitia and a well-developed neointima surrounding the retracted media were present (Fig 4ADown). Most neointimal cells exhibited the presence of the BrdU (86±5%) with staining of a variable intensity, suggesting a gradual loss of the label after multiple cell replications (Fig 4B through 4DDown).



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  		Figure 3. Porcine coronary arteries showing localization of BrdU-labeled cells 8 days after injury. A, Overview of a severely injured coronary artery (Verhoeff's stain). The adventitia is thickened in the vicinity of medial injury; early neointima blending with thrombus is present. B through D, BrdU immunostaining in the section adjacent to that shown in A. B, The edge of dissected media contains infrequent BrdU-labeled cells (brown nuclear stain) that accumulate in the adventitia and neointima. C, At the center of injury, the adventitia and neointima contain BrdU-positive cells almost exclusively. Note a high number of labeled cells infiltrating the EEL and thrombus in the process of translocation from the adventitia to neointima. D, Opposite to medial disruption, BrdU-positive cells accumulate in the adventitia, with rare labeled cells in the media. The intact media prevents adventitial cells from translocating toward the lumen. Arrows point to the EEL. a indicates adventitia; m, media; n, neointima; and t, thrombus. Magnification x20 (A) and x200 (B through D).



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  		Figure 4. Porcine coronary arteries showing localization of BrdU-labeled cells 35 days after injury. A, Overview of a severely injured coronary artery (Verhoeff's stain). A well-developed neointima fills the gap between the edges of broken media. The adventitia exhibits thickening in the vicinity of injury. B through D, BrdU immunostaining in the section adjacent to that shown in A. B, The BrdU-positive cells (brown nuclear stain) extend from the adventitia to the neointima surrounding the edge of dissected media. The media continues to contain infrequent labeled cells. C, In the center of neointima, a high number of BrdU-labeled cells is present. Note the varying intensity of BrdU staining in neointima, with the gradient from the adventitial (right) to luminal (left) sides. D, Opposite to medial injury, BrdU-positive cells are present in the adventitia; isolated labeled cells are seen in the media. The uninjured media continues to serve as a barrier preventing adventitial cell migration. Arrows point to the EEL. a indicates adventitia; m, media; and n, neointima. Magnification x20 (A) and x200 (B through D).

In vessels exhibiting mild injury, the absence of adventitial activation was associated with rare BrdU-labeled cells (<5%) in the media at all time points. Likewise, small areas of neointima at 18 to 35 days (n=4) contained infrequent labeled cells (30±14%, P<.005 versus severe injury). Thus, the media appeared to function as a protective barrier, preventing adventitial cell activation and their subsequent migration, which resulted in attenuated neointimal formation.

Characteristics of Translocating Cells
To characterize the cells that translocated from the adventitia to neointima during vascular repair, we have determined their phenotype by assessing cytoskeletal protein markers. At 2 days after injury, the adventitia was composed of fibroblasts and blood-borne cells that differed from medial SM cells lacking {alpha}-SM actin and desmin immunostaining. In the media, the majority of BrdU-labeled cells were also devoid of {alpha}-SM actin, suggesting their nonmuscle origin (Fig 5A and 5BDownDown).



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  		Figure 5. Double immunostaining for {alpha}-SM actin and BrdU, illustrating formation of myofibroblasts in the adventitia and their translocation to neointima after coronary injury. A and B, Two days after injury, BrdU-positive cells in the adventitia adjacent to the media (A) and in the center of injury (B) are devoid of {alpha}-SM actin immunostaining. In the media, the BrdU-labeled cell is negative for {alpha}-SM actin (arrow), which contrasts with quiescent medial SM cells positive for {alpha}-SM actin (A). C and D, At 8 days, some BrdU-labeled cells acquire {alpha}-SM actin within the adventitia (C), which identifies them as myofibroblasts; most neointimal cells are positive for BrdU and {alpha}-SM actin, which suggests their adventitial origin (D). The BrdU-positive cells are identified by a brown nuclear stain, {alpha}-SM actin immunostaining is depicted by a purple cytoplasmic stain. Arrowheads point to cells positive for BrdU and {alpha}-SM actin. a indicates adventitia; m, media; and n, neointima. Magnification x760.

At 7 to 8 days, the adventitia showed areas of focal immunoreactivity with {alpha}-SM actin antibodies, indicating fibroblast differentiation to myofibroblasts. Double immunohistochemistry has shown that {approx}28% of BrdU-labeled cells in the adventitia and {approx}96% of BrdU-labeled cells in the neointima colocalized {alpha}-SM actin (Fig 5C and 5DUpUp). To exclude that the latter represented inadvertently labeled medial SM cells that then migrated to neointima, in separate experiments (n=3) BrdU administration was delayed for 72 hours after injury, again demonstrating infrequent medial cell proliferation in only 5.3±2% of cells.

At 35 days, neointimal cells continued to exhibit uniform {alpha}-SM actin immunostaining. In contrast to medial SM cells, the immunoreactivity with desmin antibodies was patchy in neointima (Fig 6Down). Double immunohistochemistry for BrdU and {alpha}-SM actin or desmin demonstrated that most cells that translocated from the adventitia to neointima showed {alpha}-SM actin but not desmin immunostaining (Fig 6Down). Accordingly, neointimal myofibroblasts exhibited a distinctive repertoire of cytoskeletal protein markers ({alpha}-SM actin positive and desmin negative), distinguishing them from medial SM cells (both {alpha}-SM actin and desmin positive), nonmigratory adventitial cells, and blood-borne inflammatory cells (both {alpha}-SM actin and desmin negative).



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  		Figure 6. {alpha}-SM actin and desmin immunostaining 35 days after coronary injury. A, Overview of {alpha}-SM actin immunostaining. The uniform pattern of {alpha}-SM actin is present in neointima. B and C, Double immunostaining for BrdU and {alpha}-SM actin in the section adjacent to that shown in A. B, In neointima, the majority of BrdU-labeled cells (brown nuclear stain) continue to colocalize {alpha}-SM actin (purple). C, In the adventitia, most BrdU-labeled cells are negative for {alpha}-SM actin. D, Overview of desmin immunostaining. Desmin is patchy within neointima, which contrasts with its uniform distribution in the media. E and F, Double immunostaining for BrdU and desmin in the section adjacent to that shown in D. E, In neointima, very few BrdU-labeled cells (dark nuclear stain) colocalize desmin (purple, arrowheads). F, In the adventitia, BrdU-labeled cells are negative for desmin. Abbreviations as in Fig 5Up. Magnification x17 (A and D) and x760 (B, C, E, and F).

Characteristics of Nontranslocating cells
Notwithstanding the process of translocation of myofibroblasts to neointima, a number of BrdU-labeled cells remained in the adventitia. At 18 to 35 days after injury, these cells were found in the periphery of the adventitia and in the vicinity of the vasa vasorum. They were devoid of both {alpha}-SM actin and desmin. Their localization and morphological features (ie, round shape, large nucleus, and small cytoplasm) were consistent with blood-borne inflammatory cells that probably were recruited into the injured adventitia.


*    Discussion
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 *Discussion
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We have previously shown the induction of adventitial fibroblast proliferation and the modulation of their phenotype to myofibroblasts after arterial injury.11 The present study demonstrated direct involvement of adventitial fibroblasts in neointimal formation after severe endoluminal injury in porcine coronary arteries. The activation of adventitial cells and their migration to the luminal surface of the injured vessels appeared critical for the ensuing neointimal formation. Myofibroblasts residing in different layers of the arterial wall exhibited distinct characteristics during the vascular repair process.

Activation of Adventitial Cells
Balloon-induced injury of the coronary arteries caused a spectrum of vascular responses. Activation of the adventitia, which included cell proliferation and adventitial remodeling, was present in the arteries with severe injury and was limited to the sites where the adventitia was exposed to the vessel lumen as a result of medial disruption. Importantly, adventitial changes were associated with the subsequent formation of neointima. In contrast, when the adventitia was protected by the injured but not disrupted media, adventitial activation and the development of neointima were markedly attenuated (see the TableUp). These observations suggest a causal relationship between the exposure of the adventitia to the vessel lumen, fibroblast activation/migration, and the subsequent neointimal formation.

Unlike the involvement of medial SM cells, activation of the adventitia has not previously been characterized during vascular response to injury. Hence, the question of whether adventitial changes represent a common mechanism involved in vascular repair or are merely a model-dependent phenomenon arises. Several lines of evidence suggest ubiquitous involvement of adventitial cells in the vascular repair process. First, because of frequent removal of the adventitia during vessel processing or the quantification of cell replication only within the media and neointima, adventitial proliferation was likely overlooked in prior studies.4 5 If the adventitia is omitted from the analysis in porcine coronary arteries, the time course of medial and neointimal proliferation is similar to that described for a well-characterized rat injury model. Second, balloon denudation of rabbit femoral arteries results in activation of the adventitia (Y.S., A.Z., unpublished results). Hence, the subsequent neointimal thickening in this model could result from not only balloon-induced medial cell migration but also concomitant activation of the adventitia and the subsequent translocation of adventitial cells. Third, the injury involving the outer layer of the vessel is critical for neointimal development. Selective adventitial injury even without endothelial denudation is associated with the formation of neointima.10 Likewise, the removal of the entire media has recently been reported to be accompanied by significant neointimal formation.21

Involvement of Myofibroblasts in Neointimal Formation
We have used the in vivo BrdU labeling method to trace migration of adventitial cells toward the lumen and their involvement in neointimal formation. A rapid clearance of BrdU from the circulation allowed us to label cells in the S phase during a narrow time window.22 23 Hence, the ensuing changes in the localization of labeled cells reflected their migration as opposed to ongoing proliferation.24 25

Blood-borne inflammatory cells (eg, macrophages), SM cells, and vascular fibroblasts should be considered potential components of the BrdU-labeled cell population. The presence of BrdU-positive cells in the vicinity of the vasa vasorum and the exposure of the adventitia to blood elements after severe injury clearly suggest some infiltration of the injured adventitia with macrophages. They likely contributed to the formation of myofibroblasts through their secreted products, as previously described in wound healing.26 27 The infiltration of adventitia with blood-borne cells notwithstanding, the acquisition of {alpha}-SM actin by some labeled cells in the adventitia at 7 to 8 days indicated that proliferating fibroblasts constituted an important cellular element during the adventitial response to coronary injury.

Although cells translocating to the neointima showed certain similarities with medial SM cells (eg, {alpha}-SM actin expression), several differences underscored their separate origins. Activated adventitial cells did not express {alpha}-SM actin or desmin at 2 days after injury, whereas SM cells present in adventitial vasa vasorum and in the injured media were strongly positive for both cytoskeletal protein markers at all times. Migration of SM cells to the adventitia also was unlikely because the translocation of BrdU-labeled cells indicated cell migration in the opposite direction. It is important to emphasize that the results of this study did not preclude migration of medial cells to neointima. The media of normal arteries contains a small number of "nonmuscle" cells.28 29 In fact, after injury most labeled cells in the media lacked {alpha}-SM actin, which likely identified them as medial fibroblasts (Fig 5AUp). Recent findings indicate that these cells closely resemble adventitial fibroblasts in regard to morphology, cytoskeletal proteins, and a high growth potential, whereas medial SM cells appear unable to undergo the phenotypic changes required for neointimal formation.30 Hence, in severe medial injury, both adventitial and medial fibroblasts could contribute to neointimal formation. After milder coronary injury, however, the origin of a small neointima remains to be further clarified.

The mechanisms regulating fibroblast migration to neointima are unknown. Adventitial fibroblasts and myofibroblasts translocated toward medial dissections, which is consistent with the chemotactant gradient of locally released platelet-derived growth factor and transforming growth factor ß1.31 32 33 Intraluminal thrombus, which contains the above-mentioned cytokines, was a typical feature of severe coronary injury. It can also be postulated that migrating cells produce extracellular matrix-degrading enzymes and plasminogen activators that facilitate their movement,34 35 which parallels a similar ability of fibroblasts to degrade connective tissue in other reparative processes after tissue damage.36

Neointimal Myofibroblasts
Myofibroblasts residing in different compartments of the vascular wall exhibited distinct characteristics. Similar to myofibroblasts in wound healing, adventitial myofibroblasts were markedly reduced at later times after vascular injury.11 13 14 This may reflect their migration to neointima, the removal by apoptosis, or the reversal to an undifferentiated fibroblast phenotype. In contrast, migratory myofibroblasts that reached the neointima have shown a sustained {alpha}-SM actin expression resembling the prolonged presence of myofibroblasts in hypertrophic scars and other pathological conditions.37 38 39 Although neointimal cells uniformly expressed {alpha}-SM actin, most cells of adventitial origin (ie, BrdU-labeled) were negative for desmin at 35 days after injury. This pattern of desmin immunostaining in the neointima has previously been interpreted as evidence that medial SM cells lose their intermediate filaments after vascular injury.40 41 42 Alternatively, our findings not only suggest distinctive signaling mechanisms within the neointima but also may reflect characteristics of unique cellular components such as myofibroblasts. It is noteworthy that a similar nonuniform distribution of desmin has been observed during reparative processes involving myofibroblasts in nonvascular tissues.43

Study Limitations
The demonstrated myofibroblast migration from the adventitia to neointima was based on the tracing BrdU-labeled cells that acquired {alpha}-SM actin after severe coronary injury. Regional differences in vascular responses to endoluminal injury or the variation in fibroblast content in various vascular beds (eg, coronary versus peripheral circulation) may modify the proposed mechanism of arterial repair. The development of newer cellular markers specific for fibroblasts or myofibroblasts, which distinguish them from surrounding SM cells, may facilitate future studies.44 It is important to underscore that the migration of adventitial cells to the neointima was likely underestimated for several reasons. First, we have selected a very early time point for the BrdU administration to avoid labeling of cells that have either migrated to the media or originated from the media. This excluded those adventitial cells that entered the cell cycle later and then migrated to the neointima. Second, neointimal cells of adventitial origin exhibited decreasing intensity of the BrdU staining, probably as a result of ongoing cell divisions, resulting in the loss of the label in some cells. Finally, because cell migration also involves nonreplicating cells,45 the BrdU method could not identify all adventitial cells involved in this process.

The findings of this study describe adventitial involvement in the vessel repair process after injury to nonatherosclerotic coronary arteries. It should be underscored that vascular responses are more complex in diseased vessels, with the underlying intima and diverse components of atherosclerotic plaque. Severe vascular injury exposing the adventitia, however, is expected to occur after several transcatheter coronary interventions, including a high-pressure stent implantation, an atherectomy, or a coronary angioplasty.46 47 48 Nevertheless, the exact role of myofibroblasts in geometric remodeling and neointimal formation remains to be determined in clinical settings.

Conclusions
Vascular injury induces proliferation and migration of adventitial fibroblasts, accompanied by their phenotypic modulation to myofibroblasts. The translocation of adventitial myofibroblasts across the EEL toward the lumen after severe medial injury brings into question the sole contribution of SM cells to neointimal formation. These data suggest the significance of myofibroblasts in vascular repair and illustrate a new mechanism whereby adventitial fibroblasts may contribute to restenosis after angioplasty.


*    Selected Abbreviations and Acronyms
 
BrdU = 5-bromo-2'-deoxyuridine
EEL = external elastic lamina
IEL = internal elastic lamina
SM = smooth muscle


*    Acknowledgments
 
This study was supported in part by NIH grant HL-55410 and a standard grant-in-aid from the American Heart Association, Florida and Delaware Affiliates, Inc. Dr Fard was supported by a fellowship grant from the American Heart Association, Delaware Affiliate, Inc. We gratefully acknowledge the technical assistance of Felicia Hayes.


*    Footnotes
 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and previously published in abstract form (Circulation. 1995;92[suppl I]:I-34).

Received December 28, 1995; revision received April 1, 1996; accepted April 15, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Schwartz SM, deBlois D, O'Brien ERM. The intima: soil for atherosclerosis and restenosis. Circ Res. 1995;77:445-465.[Free Full Text]
  2. 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]
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