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Circulation. 1996;93:340-348

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(Circulation. 1996;93:340-348.)
© 1996 American Heart Association, Inc.

Articles

Adventitial Remodeling After Coronary Arterial Injury

Yi Shi, MD, PhD; Marc Pieniek, MD; Ali Fard, MD; James O'Brien, MD ; John D. Mannion, MD; Andrew Zalewski, MD

From the Department of Medicine (Cardiology), Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Andrew Zalewski, MD, Thomas Jefferson University, 1025 Walnut St, Suite 410N, Philadelphia, PA 19107.


*    Abstract
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 *Abstract
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down arrowResults
down arrowDiscussion
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Background Intraluminal thrombus formation and medial smooth muscle (SM) cell proliferation are recognized responses of the arterial system to injury. In contrast to these well-characterized processes during vascular repair, changes involving the adventitia have been largely neglected in previous studies. Hence, the goal of this investigation was to assess the response of the adventitia to coronary arterial injury.

Methods and Results Adventitial changes in porcine coronary arteries subjected to medial injury were characterized by immunohistochemistry, histochemistry, and microscopic morphometry. The rapid development of a hypercellular response in the adventitia was evident 3 days after balloon-induced medial injury. Cell proliferation, as assessed by proliferating cell nuclear antigen immunostaining, reached the maximum level in the adventitia at 3 days, whereas at 14 and 28 days, the number of replicating cells reverted toward the baseline. The proliferating activity in the adventitia exceeded that seen in the media at all times after injury. To further define the changes in the phenotype of adventitial cells, the expression of three cytoskeletal proteins (vimentin, {alpha}-SM actin, and desmin) was characterized. Fibroblasts in normal adventitia expressed vimentin but no {alpha}-SM actin or desmin. After injury, these cells acquired characteristics of myofibroblasts expressing {alpha}-SM actin, which peaked at 7 and 14 days. Desmin expression was patchy in the adventitia, as opposed to its homogeneous distribution in medial SM cells. The modulation of fibroblast phenotype was transient, inasmuch as {alpha}-SM actin immunostaining declined at 28 days after injury, when dense, collagen-rich scar was evident within the adventitia. The above-described changes involving hypercellularity of the adventitia, myofibroblast formation, and fibrosis were associated with a significant focal adventitial thickening at 3, 7, 14, and 28 days after injury (P<.01 versus uninjured coronary arteries).

Conclusions This study demonstrates the involvement of the adventitia in the vascular repair process after medial injury. The hypercellularity of the adventitial layer, proliferation of fibroblasts, and modulation of their phenotype to myofibroblasts are associated with the development of the thickened adventitia. It is postulated that these phenomena affect vascular remodeling and may provide an important insight into the mechanisms of vascular disorders.


Key Words: adventitia • remodeling • myofibroblasts • angioplasty • restenosis


*    Introduction
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 *Introduction
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down arrowResults
down arrowDiscussion
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T he vessel wall response to injury provides the pathophysiological basis for the development of a wide spectrum of cardiovascular disorders.1 Vascular insult triggers a cascade of events in which blood-borne cells and resident mesenchymal cells modulate their phenotype in response to locally released cytokines. In particular, SMC migration and proliferation, followed by extracellular matrix synthesis, have provided the overall sequence of cellular events that contribute to the development of atherosclerosis2 and vascular restenosis.3 4 5 These processes can lead to a dramatic change in microscopic composition and dimensions of the vessel wall.

Coronary angioplasty and other transcatheter procedures induce an acute form of vascular injury whose long-term revascularization benefit is limited by restenosis. Although the pathogenesis of this process is multifactorial, the formation of neointima is common after balloon injury.6 7 Recent experimental8 9 10 and clinical observations, however,11 have questioned prior assumptions that neointima correlates with luminal renarrowing. These studies suggest that geometric remodeling due to poorly defined mechanisms is most likely involved in the loss of patency after vascular injury. Since radial dimensions of the artery after injury may depend on the intactness of its outer layer, we sought to examine the changes in the adventitia of porcine coronary arteries after medial injury. This study demonstrated that vascular injury induces profound remodeling of the adventitia, including an increase in its thickness, a transient change in the phenotype of adventitial fibroblasts, and the accumulation of extracellular matrix proteins (collagens). These changes, which were not emphasized in prior studies of vascular response to injury, may have an important functional role in the overall remodeling of the arterial wall in pathological conditions.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Animal Model
Domestic crossbred pigs (Sus scrofa, n=27) weighing from 22 to 30 kg were premedicated with aspirin (650 mg PO), atropine (1 mg IM), and nifedipine (10 mg sublingual). Anesthetics consisted of intramuscular injection of ketamine (12 mg/kg) and xylazine (2 mg/kg) supplemented with intravenous infusion of diprivan (12 mg·kg-1·h-1) throughout the experiment. The right external carotid artery was surgically exposed, and heparin (10 000 U) was administered intra-arterially. With an 8F SAL 1 guiding catheter (Medtronic Interventional Vascular, Inc), the coronary ostia were cannulated under fluoroscopic guidance and intracoronary nitroglycerin was administered (100 µg). The coronary arteries were injured by use of an oversized balloon (4.0 mm) that was inflated three times (6 to 10 atm) for 30 seconds. The number of coronary arteries analyzed for each parameter is reported as "n" values in the "Results" section. Postsurgical therapy included aspirin 325 mg PO and ampicillin 250 mg IM for the next 2 days. The animals were euthanatized with an overdose of pentobarbital (100 mg/kg IV) at the times indicated in the text. All animal experiments conformed to the position of the American Heart Association on research animal use and were in accordance with institutional guidelines.

Tissue Preparation and Histochemistry
To preserve the integrity of the adventitia and perivascular tissues, porcine coronary arteries were carefully removed in a block along with adjacent tissues (ie, the adipose tissue, myocardium), rinsed with PBS, and then immersed in HistoChoice tissue fixative (Ameresco). The arteries were sectioned into 2- to 5-mm blocks, placed in individual cassettes, and fixed for at least 5 hours in HistoChoice. Then the samples were processed in a Tissue-Tek VIP processor (Miles Inc), embedded in paraffin, and cut into 5-µm-thick sections. Next, they were placed on glass slides previously coated with Vectabond (Vector Laboratories).

The tissue sections were deparaffinized; Verhoeff's stain for elastic tissues12 was used in the representative slides from each block to identify the site of the most severe medial injury, defined as a distinct disruption of the internal elastic lamina with preserved continuity of the external elastic lamina. The specimens devoid of these criteria were excluded from further studies. Hence, all analyses were carried out using sections exhibiting comparable degrees of medial injury. Adjacent sections were examined by histochemistry, immunohistochemistry, and morphometry. To determine the cellularity of vascular lesions, hematoxylin-eosin stain was used. To characterize components of the extracellular matrix, Sirius red and Alcian blue stains were used to identify collagens and proteoglycans, respectively.13

Immunohistochemistry
The Vectastain Elite ABC system (Vector Laboratories) was used for immunohistochemistry. 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. After a washing in PBS, sections were incubated with primary antibodies for 1 hour at room temperature or 24 hours at 4°C 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); monoclonal mouse NCL-VIM-V9, recognizing intermediate filament vimentin (1:100, Novocastra); and monoclonal mouse PC10 antibody, identifying PCNA (1:200, DAKO). Next, the slides were washed and incubated with biotinylated secondary horse anti-mouse antibodies (1:2000, Vector Laboratories) for 1 hour. The sections 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.

Cell Density, Proliferation Index, and Morphometric Analysis
Cell density and cell proliferation were determined by counting total cell nuclei and PCNA-positive nuclear staining, respectively, in a minimum of 250 cells per vessel layer per field. Cellularity was expressed as number of cells per square millimeter, whereas the proliferation index reflected the percentage of PCNA-positive cells. These measurements were performed in sections demonstrating comparable degrees of medial injury.

Morphometric analyses were carried out with a computerized imaging system (Advanced Imaging Concepts, Inc). The adventitia was defined between the medial edge of the external elastic lamina (inner border) and either the edge of the adipose tissue or the myocardium surrounding coronary arteries (outer border). Since the outer border often demonstrated a smooth transition into surrounding tissues, all morphometric measurements were carried out on slides stained with Verhoeff's stain at the same magnification that provided the optimal demarcation of the adventitia. The minimal and maximal adventitial thicknesses (in micrometers) as well as medial and neointimal thicknesses were calculated. In the control vessels, measurements were carried out on multiple sections (three or four per vessel) to account for naturally occurring variability in medial and adventitial dimensions. In injured coronary arteries, morphometric measurements were carried out in the sections demonstrating the most severe signs of medial injury to capture the maximal response. To minimize error of measurements, each parameter was calculated three times, and the average value was reported. The intraobserver variability for repeat measurements was <10%.

Statistical Methods
All numerical data are presented as mean±SEM. One-way ANOVA was used to compare the time-dependent variables. If the F test results were significant, Bonferroni 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 Thickening After Injury
The thickness of the adventitia in normal porcine coronary arteries varied from 131±6 µm (minimum diameter) to 224±9 µm (maximum diameter) in individual sections, with mean values of 178±44 µm (n=9). To assess adventitial remodeling after vascular injury, the maximal adventitial diameter was measured at 3 (n=4), 7 (n=4), 14 (n=4), and 28 (n=6) days after balloon injury and was compared with the maximal adventitial diameter of normal coronary arteries. Adventitial thickness was significantly increased at all time points compared with normal vessels (P<.01, Fig 1Down). As depicted in Fig 2Down, the most striking changes in adventitial dimensions were present in the regions adjacent to the site of medial injury. In contrast, the thickness of the adventitia opposite medial injury or in other uninjured sections from the same vessels remained largely unaffected.



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  		Figure 1. Bar graph demonstrating maximal adventitial, medial, and neointimal thickness (µm). An increase in maximal adventitial diameter is observed at 3, 7, 14, and 28 days after coronary arterial injury compared with baseline, ie, uninjured coronary arteries (P<.01), whereas medial diameter remains unchanged. As expected, neointimal thickness continues to increase from day 7 after injury.



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  		Figure 2. Photomicrographs showing adventitial remodeling in porcine coronary arteries after balloon injury. A and B, At 3 days, adventitial thickening is circumferential, involving both the injured (A) and the opposite (B) sides of the vessel. C and D, At 28 days, an increase in the adventitial thickness is more striking in the vicinity of medial injury (C) compared with the opposite site of the same section (D). A and C show vascular remodeling directly adjacent to medial injury, whereas B and D exhibit the adventitia and the intact media on the opposite side of the same sections, respectively. Arrows point to adventitial borders. a indicates adventitia; m, media; and n, neointima. Verhoeff's stain, magnification x25.

Cellular Response in the Adventitia After Injury
A deep medial injury without disruption of the external elastic lamina was associated with an increase in adventitial cell density beginning at 3 days, which returned to baseline at 14 days. In control (ie, uninjured) coronary arteries, adventitial cell density was 3880±372 cells/mm2 (n=5), which increased to 7094±576 cells/mm2 at 3 days (n=4, P<.01) and 7218±256 cells/mm2 at 7 days (n=4, P<.01) after injury. At 14 and 28 days, cell density in the adventitia was 4617±208 cells/mm2 (n=4, P=NS versus controls) and 4989±547 cells/mm2 (n=6, P=NS versus controls), respectively, returning toward baseline (Fig 3Down). The segments of coronary arteries remote from the site of vascular injury resembled uninjured vessels exhibiting no hypercellular response at all time points.



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  		Figure 3. Graph showing cell density in the adventitia. In regions adjacent to the site of medial injury, a significant increase in cell density is observed at 3 and 7 days after balloon injury compared with baseline (ie, uninjured vessels, P<.01).

The increase in the cellularity of the adventitia was paralleled by high proliferative activity. With PCNA staining, replicating cells were identified in 3±1% of adventitial cells in control coronary arteries (n=4), whereas at 3 (n=3) and 7 (n=3) days after injury, the proliferating index was significantly higher at 42±6% (P<.01) and 34±4% (P<.01), respectively. As depicted in Fig 4Down, this proliferative response in the adventitia exceeded values observed in the media after balloon injury. At 14 (n=4) and 28 (n=3) days after medial injury, the adventitia was largely quiescent, with 2±1% of cells expressing PCNA at each time (P=NS versus controls, Fig 4Down). As illustrated in Fig 5Down, at 3 days after injury, proliferating cells were circumferentially distributed in the adventitia, with fewer PCNA-positive cells present within the media. A similar distribution of actively dividing cells was observed with 5-bromo-2'-deoxyuridine labeling (data not shown). At later time points, PCNA-positive cells accumulated predominantly in the portion of the adventitia in the vicinity of medial injury as well as in the newly formed neointima.



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  		Figure 4. Bar graph demonstrating proliferation index within the adventitia and media. Cell proliferation is significantly increased within the adventitia and media at 3 and 7 days after coronary injury compared with baseline (ie, uninjured vessels, P<.01). Note higher level of cell proliferation in the adventitia than in the media at all times after injury.



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  		Figure 5. . Photomicrographs demonstrating cell proliferating activity (PCNA staining) in the adventitia after coronary balloon injury. A, At 3 days, striking cell proliferation is evident. The majority of positive cells (brown nuclear stain) are located within the adventitia. Note medial injury with thrombus. B, Opposite side of same section shown in A. Note intact media, with the majority of PCNA-positive cells localized in the adventitia. PCNA-positive cells in the media are present at the edges of medial dissection (A) and in deeper layers of the media (B). C, At 28 days, cell proliferation is infrequent in the thickened adventitia. Few positive cells are visible within neointima. D, Opposite side of same section as shown in C. Note low proliferating activity within media and adventitia. Arrows point to the external elastic lamina, representing the inner border of the adventitia. a indicates adventitia; m, media; n, neointima; and t, thrombus. Magnification x25.

Cellular Composition in the Adventitia After Injury
To identify the cellular composition in the adventitia, vascular specimens were subjected to immunohistochemistry, with monoclonal antibodies recognizing major cytoskeletal proteins of mesenchymal cells (n=3 to 5 vessels per time point). In uninjured coronary arteries, adventitial cells were uniformly positive for vimentin but negative for {alpha}-SM actin and desmin (V type, not shown). In contrast, medial SMCs showed strong immunoreactivity, with antibodies against all three cytoskeletal proteins (VAD type). Coronary arterial injury did not alter vimentin expression, but it did increase the {alpha}-SM actin and desmin expression in the adventitia. The adventitia containing hypercellular, granulation-like tissue exhibited weakly positive staining with {alpha}-SM actin antibodies at 3 days. The immunostaining for {alpha}-SM actin became strongly positive within the adventitia at 7 and 14 days after injury (VA type, Fig 6Down). This change in the phenotype of adventitial fibroblasts to myofibroblasts (ie, containing {alpha}-SM actin) was particularly evident in the areas adjacent to medial injury, although circumferential localization of these cells was occasionally noted at 7 days. There was no evidence for {alpha}-SM actin immunostaining in the adventitia beyond the site of medial injury. As shown in Fig 6Down, the presence of myofibroblasts appeared to decline at later times, with frequent disappearance at 28 days.



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  		Figure 6. {alpha}-SM actin immunostaining showing myofibroblast formation in the adventitia of coronary arteries after balloon injury. A and B, At 3 days: weakly positive {alpha}-SM actin immunostaining is visible in the adventitia adjacent to medial injury (A); no {alpha}-SM actin immunoreactivity is present on the opposite side (B). C and D, At 7 days: strong {alpha}-SM actin immunostaining is evident in the vicinity of medial injury (C); note less dense myofibroblast presence on the opposite side (D). E and F, At 28 days: {alpha}-SM actin immunostaining becomes attenuated. A, C, and E represent the thickened adventitia directly adjacent to medial injury, whereas B, D, and F show the adventitia and the intact media on the opposite side of the same sections, respectively. m indicates media; a, adventitia. Magnification x25.

In general, the time course of immunoreactivity with desmin antibodies in the adventitia paralleled that of {alpha}-SM actin (VAD type). However, desmin-positive cells were less frequent, with more patchy distribution in the adventitia after medial injury (Fig 7Down).



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  		Figure 7. Desmin immunostaining in the adventitia of coronary arteries after balloon injury. A and B, At 3 days: the adventitia is devoid of desmin. C and D, At 7 days: weak desmin immunostaining is evident in the vicinity of medial injury (C); note its absence on the opposite side (D). E and F, At 28 days: desmin immunostaining is largely absent. Focal positive staining identifies increased vasa vasorum in the thickened adventitia. Note the paucity of vasa vasorum in the adventitia opposite the site of injury. A, C, and E represent the thickened adventitia directly adjacent to medial injury, whereas B, D, and F exhibit the adventitia and the intact media on the opposite side of the same sections, respectively. m indicates media; a, adventitia. Magnification x25.

Extracellular Matrix Deposition in the Adventitia
The above changes leading to myofibroblast formation were associated with a striking accumulation of collagen-containing scar in the thickened adventitia by Sirius red (Fig 8Down) and Masson's trichrome (not shown) histochemical staining. In contrast, proteoglycans were mostly confined to the neointima at 28 days after injury (Fig 8Down).



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  		Figure 8. Histochemical stains identifying major components of the extracellular matrix in the adventitia at 28 days after balloon injury. A, Fibrillar collagens by Sirius red stain; B, proteoglycans by Alcian blue stain. Note thickened adventitia containing mostly collagens (red), whereas proteoglycans (blue) are mostly confined to the neointima. Arrowheads point to the thickened adventitia. m indicates media; n, neointima. Magnification x5.


*    Discussion
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*Discussion
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This study indicates that adventitial fibroblasts/myofibroblasts are involved in the vascular repair process, resulting in significant cellular and structural changes affecting the adventitia of injured coronary arteries.

Proliferation of Adventitial Fibroblasts
Tissue response to injury involves a cascade of adaptive phenomena that were evolutionarily developed to close an open wound.14 15 16 There are striking similarities between the process of wound healing and the response of the arterial wall to injury.17 They involve the disruption of tissue continuity as well as a chain of interconnected events allowing cells to assume "new" functions according to microenvironmental needs. Proliferation of medial SMCs has been considered a common event shortly after vascular injury.3 4 18 However, as shown in this study, adventitial rather than medial cell proliferation was typical shortly after coronary arterial injury. This process reached a maximum at 3 days, when few medial SMCs were replicating. Hypercellular adventitia after coronary injury contained vimentin, a known marker of mesenchymal cells,19 which identified these cells as fibroblasts. It is important to emphasize that the initial paucity of {alpha}-SM actin in the adventitia distinguished these cells from medial SMCs. Notwithstanding the above, macrophages are probably also present in injured adventitia during an early phase of vascular repair, inasmuch as they may express vimentin (as opposed to other blood-borne cells).20

Phenotypic Modulation of Adventitial Fibroblasts
In normal coronary arteries, vimentin-rich adventitial fibroblasts (V type) can easily be distinguished from medial SMCs, which exhibit positive staining not only with vimentin antibodies but also with antibodies recognizing {alpha}-SM actin and desmin (VAD type).21 22 Shortly after medial injury (ie, within 3 days), the adventitia becomes hypercellular with concomitant significant proliferative activity that resembles the formation of granulation tissue containing replicating fibroblasts in wound healing. The change in the phenotype of adventitial fibroblasts to myofibroblasts is reflected by the induction of {alpha}-SM actin, reaching a maximum at 7 and 14 days after injury (Fig 6Up, VA type), with some myofibroblasts also acquiring desmin (Fig 7Up, VAD type). The mechanism(s) underlying the above process of phenotypic modulation in vascular tissue remains to be determined. However, it is noteworthy that transforming growth factor-ß1 has been implicated in the induction of {alpha}-SM actin expression in wound myofibroblasts.23 24

There are several potential explanations for the disappearance of myofibroblasts noted at later times after vascular injury (Fig 6Up). The possibility of their migration to the luminal surface, which may contribute to the formation of neointima, should be considered.25 In fact, direct adventitial injury has been demonstrated to produce neointimal lesions even without endothelial denudation in several experimental models.26 27 28 29 The difficulty in ascertaining the contribution of myofibroblasts to neointimal formation, however, lies in the similarities between myofibroblasts (VA and VAD types) and SMCs (VAD type) in regard to their morphology and the spectrum of cytoskeletal protein expression. Although these cells demonstrate opposite changes in {alpha}-SM actin expression, with a reversible switch from {alpha}-SM actin to other actin isoforms in SMCs and adventitial cells acquiring {alpha}-SM actin after injury, {alpha}-SM actin never completely disappears in medial cells.30 The apoptotic cell death may represent another mechanism removing myofibroblasts from the adventitia, as it has been involved in the elimination of mesenchymal cells in dermal wounds and in restenotic lesions.16 31 The regression to fibroblast phenotype is also possible, since the reactivation of {alpha}-SM actin expression can be elicited with vessel reinjury (data not shown).

Role of Adventitial Injury
The transition of fibroblasts to myofibroblasts (ie, positive for {alpha}-SM actin) is associated with several biological activities, including enhanced collagen synthesis32 33 34 and tissue contraction/retraction, which is often associated with scar formation.16 35 Accordingly, the formation of hypercellular, myofibroblast-rich adventitia that is subsequently replaced by dense, collagen-rich scar tissue may have important implications with regard to early and late events in vascular repair. The expression of contractile cytoskeletal proteins in myofibroblasts, in particular {alpha}-SM actin, has been a hallmark of collagen matrix gel remodeling in vitro36 and various fibrocontractive disorders in vivo.32 37 38 39 Hence, vascular tissue contraction may represent a putative mechanism of vessel constriction that has recently been reported to correlate with residual stenosis after experimental angioplasty.10 Unfavorable geometric remodeling has been documented after balloon injury in several models of experimental angioplasty,8 9 10 although it has not been found by others.40

The deposition of collagen in the adventitia, as demonstrated by histochemical staining in this study (Fig 8Up), is consistent with the reported transcriptional activation of fibrillar procollagen genes after experimental angioplasty.41 Procollagen {alpha}1(I) and {alpha}1(III) mRNA levels increase between 2 and 7 days after injury, with collagen becoming the most abundant protein, constituting >50% of the arterial proteins at 30 days.41 Accordingly, deposits of fibrillar collagens may contribute to the formation of a stiff, "collarlike" adventitia that prevents coronary arteries from undergoing compensatory dilatation during neointimal formation, typical of the adaptive changes during the slow growth of an atherosclerotic plaque.42 43

Clinical Implications
Recent advances in interventional cardiology have led to more aggressive strategies to relieve coronary obstruction and often to ablate the underlying atherosclerotic plaque. Thus, a deep medial injury that may potentially affect the adventitia appears to be common in clinical practice.44 The possibility of myofibroblast formation and the deposition of extracellular matrix in the adventitia after coronary arterial injury in humans may lead to vascular tissue retraction, with the possible exception of intracoronary stenting. In fact, recent findings with intravascular ultrasound appear to corroborate this possibility, inasmuch as patients with coronary restenosis after angioplasty exhibit a smaller vessel circumference along the external elastic lamina, which delineates the adventitial border.11

The failure of many pharmacological approaches to reduce restenosis in clinical settings has stimulated considerable interest in a site-specific therapy after coronary angioplasty.45 The involvement of the adventitia in the vascular repair process may require the development of strategies allowing for the administration of potentially active compounds not only to the media but also to the outer layers of the vessel wall. This clearly increases the complexity of local drug delivery in diseased, atherosclerotic vessels, since the possibility of additional vascular trauma is of concern with more aggressive approaches.

Conclusions
This study demonstrates the involvement of the adventitia in the vascular repair process in the coronary vasculature in a porcine model. The hypercellularity of the adventitial layer due to proliferation of fibroblasts was seen early after coronary arterial injury (3 to 7 days). The expression of {alpha}-SM actin was evident in abundant adventitial myofibroblasts at 7 and 14 days. This was followed by the accumulation of collagen-containing scar tissue within the adventitia. These changes were accompanied by focal thickening of the outer layer of the coronary arteries. Hence, the adventitial response contributes to vascular remodeling after arterial injury.


*    Selected Abbreviations and Acronyms
 
PCNA = proliferating cell nuclear antigen
SM = smooth muscle
SMC = smooth muscle cell
VAD = positive for vimentin, {alpha}-SM actin, and desmin


*    Acknowledgments
 
This study was supported in part by a standard Grant-in-Aid from the American Heart Association, Florida and Delaware Affiliates, Inc. The authors gratefully acknowledge the technical assistance of Dian Wang and Carolyn Talbot.

Received July 18, 1995; revision received September 7, 1995; accepted September 11, 1995.


*    References
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up arrowResults
up arrowDiscussion
*References
 

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