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Circulation. 1996;93:2178-2187

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(Circulation. 1996;93:2178-2187.)
© 1996 American Heart Association, Inc.

Articles

Identification of a Potential Role for the Adventitia in Vascular Lesion Formation After Balloon Overstretch Injury of Porcine Coronary Arteries

Presented at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 15, 1994, and at Experimental Biology 95, Atlanta, Ga, April 12, 1995, and published in abstract form (Circulation 1994;90[pt 2]:I-392 and FASEB J 1995;9:A845).

Neal A. Scott, MD, PhD; Gustavo D. Cipolla, DVM; Cheryl E. Ross, BS; Bradley Dunn, BS; Francis H. Martin, PhD; Lizette Simonet, PhD; Josiah N. Wilcox, PhD

From the Department of Medicine, Emory University, Atlanta, Ga (N.A.S., G.D.C., C.E.R., B.D., J.N.W.) and Amgen Pharmaceuticals Inc, Thousand Oaks, Calif (F.H.M., L.S.).

Correspondence to Josiah N. Wilcox, PhD, Emory University, Box AJ, Hematology, 1639 Pierce Dr, Room 1115 WMRB, Atlanta, GA 30322. E-mail medjnw@emory.edu.


*    Abstract
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Background In the present series of experiments, we examined the onset of cell proliferation and growth factor expression after balloon overstretch injury to porcine coronary arteries.

Methods and Results Domestic juvenile swine underwent balloon overstretch injury to the left anterior descending and circumflex coronary arteries with standard percutaneous transluminal coronary angioplasty balloon catheters. To identify proliferating cells, 5-bromo-2-deoxyuridine (BrDU) was administered over a period of 24 hours before the animals were killed at either 1, 3, 7, or 14 days after injury. Immunohistochemistry was performed with monoclonal antibodies to BrDU and smooth muscle cell markers. Three days after injury, a large number of proliferating cells were located in the adventitia, with significantly fewer positive cells found in the media and lumen. Seven days after injury, proliferating cells were found primarily in the neointima, extending along the luminal surface. In situ hybridization for PDGF A-chain and ß-receptor mRNAs revealed that the expression of these two genes was closely correlated with the sites of proliferation at each time point. Studies in which BrDU was injected between days 2 and 3 and the animals were killed on day 14 suggested that the proliferating adventitial cells may migrate into the neointima.

Conclusions These data suggest that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors, and possibly migrating into the neointima. Increased synthesis of {alpha}-smooth muscle actin observed in the adventitial cells after arterial injury may constrict the injured vessel and contribute to the process of arterial remodeling and late lumen loss after angioplasty.


Key Words: adventitia • angioplasty • restenosis • muscle, smooth • remodeling


*    Introduction
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 *Introduction
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Percutaneous transluminal coronary angioplasty has become increasingly important in the management of coronary artery disease; however, restenosis in 30% to 40% of PTCA patients limits its usefulness.1 The pathogenesis of postangioplasty restenosis has not been well defined. The lesion is composed largely of SMCs and develops within 3 to 6 months. Morphological observations assembled from human vessels after PTCA have been primarily descriptive, with few insights into causes or the time course of the development of the postangioplasty restenosis lesion.2 3 Although cellular proliferation after balloon injury is the most likely mechanism for the development of clinical postangioplasty restenosis, there is controversy regarding the degree of the proliferative response. This work has relied on the examination of biopsies from postangioplasty restenosis lesions obtained by subsequent directional atherectomy and proliferating cell nuclear antigen staining to determine the extent of SMC proliferation. These studies have yielded conflicting results, with some studies suggesting a high rate of SMC proliferation, >15%,4 while others describe a more modest rate of proliferation in these arteries, 0% to 1%,5 approaching that seen in primary atherosclerotic lesions.6

There is considerable evidence that endothelial denudation of peripheral vessels in combination with various degrees of SMC damage in rats and rabbits induces SMC proliferation and development of a neointima. In these models, SMC proliferation begins in the media within 48 hours after injury, and after 1 week, these cells migrate across the internal elastic lamina to form an intimal mass of actively proliferating cells.7 8 Subsequently, the vascular lesion is formed from SMC proliferation and matrix synthesis, which continues in the intima for as long as 3 months after injury. However, the rat and rabbit may not be good models for the development of drugs to prevent human postangioplasty restenosis. Calcium channel blockers,9 heparin,10 cilazapril,11 and other agents that effectively reduce intimal lesion formation in injured rat or rabbit vessels have been ineffective in clinical trials to prevent postangioplasty restenosis.12 13 14

Pigs have been used as models for postangioplasty restenosis with some success.15 16 17 18 19 20 21 22 Injury of porcine coronary arteries either by PTCA using an oversized balloon catheter or placement of an overexpanded percutaneously delivered tantalum wire coil stimulates the formation of vascular lesions morphologically similar to those seen in human postangioplasty restenosis.16 18 A major advantage of the porcine model is the ability to study coronary arteries rather than peripheral vessels. Since the coronary arteries of the pig are similar in size to human vessels, the same catheters and protocols can be used as are used in clinical PTCA.

In the present study, we have undertaken an examination of the time course of cell proliferation and growth factor synthesis after balloon overstretch injury of porcine coronary arteries to better understand the mechanism of lesion formation in this model. Domestic juvenile swine underwent injury to the left anterior descending and circumflex coronary arteries with standard PTCA balloon catheters. To identify proliferating cells, BrDU was administered before the animals were killed and was localized by immunohistochemistry. In situ hybridization for PDGF A-chain, PDGF {alpha}-receptor, and PDGF ß-receptor mRNAs was performed with 35S-labeled porcine-specific riboprobes.


*    Methods
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Injury of Pig Coronary Arteries
Normal juvenile female domestic Yorkshire swine (n=16; weight range, 25 to 35 kg) were immobilized by an intramuscular injection of ketamine (25 mg/kg)/acepromazine (0.1 mL/kg) and atropine (0.06 mg/kg). Brevital was then administered (10 mg/kg IV), and general anesthesia was maintained with 1% to 2% isothane. An 8F sheath (generous gift of USCI) was inserted into the right femoral artery, and an 8F guide catheter (generous gift of SciMed) with a hockey-stick curve was advanced to the ostium of the left coronary artery under fluoroscopic guidance. After administration of nitroglycerin (0.6 µg/kg IC) and angiography to estimate the size of the vessel, balloon injury was then performed on the left anterior descending, circumflex, or right coronary artery with clinical PTCA catheters (generous gift of Advanced Catheter Systems and Cordis Corp) sized so that the ratio of the inflated balloon to artery was {approx}1.3:1.0. The balloon was inflated to 10 atm for 30 seconds with a 1-minute rest period followed by inflation at the same site for a total of three 30-second inflations. The catheters were withdrawn, the cutdown site was sutured, and the animals were allowed to recover from the procedure. Aspirin was administered daily (325 mg) to each animal until they were killed. Animal studies were approved by the Emory University Institutional Committee for the Care and Use of Animals and were in accordance with federal guidelines.

BrDU Injection
BrDU (Sigma Chemical Co) was dissolved in sterile lactated Ringer's solution (33 mg/mL) and administered via the ear vein in three doses of 50 mg/kg at 24, 16, and 8 hours before necropsy. The animals were killed with an overdose of barbiturate 1 day (n=2), 3 days (n=4), 7 days, (n=5), or 14 days (n=5) after angioplasty, the heart was rapidly removed, and the left coronary artery was perfused with saline to clear the blood. The injured coronary artery segment was removed in block fashion and fixed by immersion in 4% paraformaldehyde in NaPO4 buffer (pH 7.4).

Immunohistochemistry
BrDU-containing cells were detected in tissues by a specific BrDU monoclonal antibody (Dako; 1/20 dilution) after predigestion of the tissue with proteinase K (1 µg/mL) and 4N HCl. BrDU immunohistochemistry was performed with biotinylated horse anti-mouse IgG (Vector; 1/400 dilution) and the ABC-AP kit either with Vector red and counterstaining with hematoxylin for quantification or with Vector blue without counterstaining for photomicrographs, as described by the manufacturer (Vector Laboratories).

The proliferating cells were identified with the following primary antibodies (all obtained from Sigma): SM-1 directed against {alpha}-smooth muscle actin23 (1/800 dilution), smooth muscle myosin24 (1/1000 dilution), h-caldesmon24 (1/1000 dilution), vimentin25 (1/320 dilution), or desmin26 (1/10 dilution). The myosin antibody used has been shown to be specific for SMCs and reacts with smooth muscle myosin heavy chain of 204 and 200 kD but does not cross-react with skeletal, cardiac, or nonmuscle myosins. The h-caldesmon antibody is specific for the 120- to 150-kD h-caldesmon and does not cross-react with skeletal or cardiac muscle or with the 70-kD nonmuscle caldesmon and has also been shown previously to preferentially stain SMCs.24 These antibodies were used in either single- or double-label immunohistochemistry to aid in the identification of the proliferating cells.

Single-label immunohistochemistry was performed with ABC-peroxidase as described by the manufacturer (Vector Laboratories). Endogenous peroxidase was blocked with 0.3% H2O2/methanol, and the slides were incubated at room temperature with the primary antibodies at the indicated dilutions for 1 hour followed by washing in PBS, incubation with a 1/400 dilution of biotinylated horse anti-mouse IgG (Vector), and finally staining with the ABC-peroxidase kit and diaminobenzidine with hematoxylin counterstaining.

Double-label immunohistochemistry was performed by staining first with the antibody directed against BrDU as described. The tissues were then washed in PBS, followed by 10 minutes of incubation with biotin blocking solution (Vector Laboratories), a PBS wash, dehydration in graded alcohols, and treatment with Americlear (Baxter Diagnostics, Inc), and then were air-dried. Endogenous peroxidase was blocked with 0.3% H2O2/methanol, and the slides were treated with 1% gelatin in PBS and exposed to the second primary antibody overnight at room temperature. The next day, the slides were washed in PBS and incubated with a 1/400 dilution of biotinylated horse anti-mouse IgG (Vector) and stained with the ABC-peroxidase kit. The slides were dehydrated in graded alcohols and Americlear and coverslipped without counterstaining to enhance the contrast. Similar results were observed with single-antibody staining in every case. Some slides were counterstained with hematoxylin for the purposes of quantifying the extent of double staining.

The single- and double-label immunohistochemistry experiments were controlled by either elimination of the primary antibody or incubation of the tissue with a nonimmune mouse IgG. No staining was ever observed in either case, confirming the specificity of the staining reactions.

Image Analysis
Color video images of 280x360-µm fields were captured and digitized by use of a x25 objective with a Sony DXC-760MD video camera, a RasterOps 24 XLTV video card, and Media Grabber software on a Macintosh Quadra 950 computer. The digital images were then analyzed by splitting of the color images into their red and blue components for the determination of blue (hematoxylin-positive) and red (BrDU-positive) cells, respectively, by use of the IP Lab Spectrum software package (Signal Analytics Corp). Positive and negative cells of each color were differentiated by setting threshold values and cell size discriminators that yielded the best identification of positive cells as judged by the operator. Each analysis was subjected to critical examination by a blinded operator, and cells were added or removed from the computer count to accurately reflect the number of red or blue cells in the microscope field. This analysis has been validated by comparison to manual counts alone and yields essentially the same results. Ten vessels were analyzed by independent observers using manual or computer counting methods. There was a significant correlation between the percentage of BrDU-positive cells determined by manual counting (9.41±2.01%) compared with the computer counting method (9.88±1.35%) (r=.892; P=.0005). Consistency was determined throughout the computer analysis by repeated analysis of five control fields from a control BrDU-labeled vessel that showed a variation of <3%.

Data obtained from both the left anterior descending and circumflex arteries were combined. Two cross sections from each vessel {approx}3 mm apart were stained and counted. Cell proliferation was analyzed at x250 magnification in five regions in each vessel as follows: Region 1, in the media adjacent to the medial tear; Region 2, in the media on the side opposite the medial tear; Region 3, in the adventitia adjacent to the medial tear; Region 4, in the adventitia on the side opposite the medial tear; and Region 5, in the intima defined as the luminal side of the external elastic lamina between the torn ends of the media (Fig 1Down). All of the cells in each region were counted, meaning that depending on the size of the vessel, two to four fields at x250 magnification were captured and analyzed in each region. The percent of proliferating cells (total number of BrDU-labeled cells divided by total number of hematoxylin-labeled cells times 100) in each region was averaged over the number of fields and the number of cross sections examined for each vessel. The vessel means, determined for each region of each vessel at each time point, were then used as individual data points for statistical comparison. Only those vessels with distinct medial tears corresponding to the injury classification II to III (a clear break in the internal elastic lamina and media with a 25% to 50% gap in the media without compromising the external elastic lamina) as previously described19 were analyzed. This eliminated from the study those vessels in which the balloon catheter failed to break the internal elastic lamina and media (8 vessels) or in which multiple fractures of the media were found (4 vessels). Thrombus formation was not a significant feature in these vessels, and only 30% of the vessels examined (6/20 of the 1-, 3-, and 7-day specimens) had small platelet thrombi on the luminal surface, whereas hemorrhage behind the medial dissection into the adventitial space localized to the region of injury was seen consistently in all vessels examined.



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  		Figure 1. Summary of regions used for analysis of cell proliferation by BrDU immunohistochemistry and computer-assisted image analysis. Cell proliferation was analyzed at x250 magnification in five regions in each vessel: Region 1, in the media adjacent to the medial tear; Region 2, in the media on the side opposite the medial tear; Region 3, in the adventitia adjacent to the medial tear; Region 4, in the adventitia on the side opposite the medial tear; and Region 5, in the intima defined as the luminal side of the external elastic lamina between the torn ends of the media. Due to the lack of a neointima at earlier time points, Region 5 was analyzed only on days 7 and 14. This is a cross section of a porcine left anterior descending coronary artery 14 days after balloon overstretch injury with a clinical PTCA balloon catheter. Arrows mark the ends of the broken internal elastic lamina. Magnification x10.

To determine the amount of BrDU staining in the neointima at day 14 resulting from an injection of BrDU between days 2 and 3, tissue sections were stained with the BrDU antibody alone and counterstained with hematoxylin. Eight random fields were counted manually from two cross sections from each animal at x250 total magnification, and the number of BrDU-positive and the total number of hematoxylin-stained cells were determined in each. It was not possible to get an accurate determination of the number of BrDU-positive cells with the computer-based system because there was a significant variation in the intensity of labeling of these cells, presumably because many of these cells had lost labeled DNA as a result of the number of times they replicated since injection of BrDU.

In Situ Hybridization
In situ hybridization was performed on frozen tissue sections with porcine-specific 35S-labeled sense and antisense riboprobes as described.27 Porcine cDNA fragments encoding for PDGF A chain (492 bp), PDGF {alpha}-receptor (670 bp), and PDGF ß-receptor (407 bp) were amplified from porcine SMCs and subcloned into pCRII vectors (Invitrogen). The following primer sequences were used for PCR amplification: PDGF A chain, 5'-AGCATCCAGCGCCTCGGGAC, 3'-CAGTTCCACCGGTTCCACCTCA; PDGF ß-receptor, 5'-GGACTTCCTGGAGGGGGTGA, 3'-CGTTTTGGTGGTAACCCCTGTCCC; PDGF {alpha}-receptor, 5'-GCIIAATAACITCGGAGGAGAAGT, 3'-ATGTAGATACACGGCCTGGGC. These cDNAs were sequenced except for a small portion of PDGF {alpha}-receptor and showed 90.4%, 88.4%, and 85.2% homology with human PDGF A chain, PDGF {alpha}-receptor, and PDGF ß-receptor, respectively.

Statistics
The numbers of proliferating cells as determined by BrDU immunohistochemistry and computer-aided image analysis were compared by one-way ANOVA and the Tukey-Kramer multiple comparisons test (Instat version 2.01, GraphPad Software).


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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No proliferating cells were found in normal uninjured vessel segments, nor were proliferating cells found in any compartment, 1 day after PTCA (TableDown). Examination of BrDU staining on day 3 revealed a large number of proliferating cells in the medial wall and the adventitia. Few positive cells were found in region 1 in the media at the site of the medial tear at this time (10.53±1.34%), and many of these were actually found on the luminal surface and probably represent proliferating endothelial cells. Fewer BrDU-positive cells were found in the media in region 2 on the side opposite the medial tear (6.42±0.74%). A large number of proliferating cells were distributed circumferentially around the vessel in the adventitia extending from region 3, at the site of the medial tear (27.34±2.32%), to region 4, on the opposite side of the vessel (16.77±4.24%).


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  		Table 1. Time Course and Distribution of Cell Proliferation After Balloon Overstretch Injury of Porcine Coronary Arteries

Seven days after PTCA, proliferating cells were found mainly in the neointima in region 5 (19.21±3.66%). Proliferating intimal cells were often found along the luminal surface, with a few proliferating cells in the adjacent media (2.78±0.68%). Relatively few BrDU-positive cells were found in the adventitia or in the media away from the lesion site at this time compared with day 3. Few proliferating cells were found in any compartment 14 days after injury; however, if proliferating cells were present, they were found primarily in the neointima (7.45±1.53%).

In situ hybridization for PDGF A chain and ß-receptor revealed that the expression of these two genes was closely linked with cell proliferation. There was no production of PDGF A-chain or PDGF ß-receptor mRNA in uninjured vessels. Three days after injury, the predominant site of PDGF A-chain expression was in the adventitia adjacent to the vessels (Fig 2Down). This was not restricted to the adventitia at the break site but rather extended circumferentially around the arteries in the same pattern as the BrDU staining. PDGF ß-receptor expression corresponded to that seen with PDGF A chain, with a large increase in expression in the adventitia 3 days after injury. Although a few PDGF A-chain and ß-receptor mRNA–positive cells were found in the media adjacent to the tear, the predominant site of mRNA localization was in the adventitia.



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  		Figure 2. Cell proliferation and growth factor expression 3 days after balloon overstretch injury of porcine coronary arteries. The distribution of cells containing PDGF A-chain (A), PDGF ß-receptor (B), and PDGF {alpha}-receptor (C) mRNAs is compared with the distribution of proliferating cells (D) as determined by injection of BrDU between days 2 and 3 after angioplasty and immunohistochemistry using a BrDU-specific antibody on serial 5-µm frozen tissue sections. Note the localization of PDGF A-chain and ß-receptor mRNAs in proliferating cells in the adventitia and the lack of PDGF A-chain mRNA and cell proliferation in the medial wall cells. PDGF ß-receptor expression was increased throughout the media, even extending circumferentially to the media on the side opposite the medial tear (not shown). PDGF A-chain mRNA was not detected in the medial SMCs. PDGF {alpha}-receptor mRNA was not detected in these tissues but served as a useful negative control. All the in situ slides shown were exposed for 8 weeks. Magnification: A through C, x80; D, x40. Break in the medial wall is indicated by the arrows. EEL indicates external elastic lamina.

One week after angioplasty, PDGF A-chain expression was found primarily in the neointima along the luminal surface of the developing lesion colocalized with PDGF ß-receptor at sites of cell proliferation, as indicated by BrDU staining (Fig 3Down). PDGF ß-receptor expression continued to be elevated in the adventitia and in scattered cells in the medial wall near the site of medial injury, whereas PDGF A-chain mRNA was not detected in these tissues at this time. This was most apparent in the medial wall opposite the break site of the vessel, where PDGF ß-receptor continued to be expressed 1 week after injury without corresponding PDGF A-chain mRNA localization (data not shown).



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  		Figure 3. Cell proliferation and growth factor expression 7 days after balloon overstretch injury of porcine coronary arteries. The distribution of cells containing PDGF A-chain (A), PDGF ß-receptor (B), and PDGF {alpha}-receptor (C) mRNAs is compared with the distribution of proliferating cells (D) as determined by injection of BrDU between days 6 and 7 after angioplasty. Note the localization of PDGF A-chain and ß-receptor mRNAs in proliferating cells in the neointima. PDGF ß-receptor expression remained elevated throughout the media, whereas PDGF A-chain mRNA was detected only in the neointima. Cell proliferation or growth factor expression was not found in the adventitia at this time (not shown). PDGF {alpha}-receptor mRNA was not detected in these tissues. All the in situ slides shown were exposed for 8 weeks. Magnification: A through C, x80; D, x40. Break in the medial wall is indicated by the arrows. EEL indicates external elastic lamina.

Neither PDGF A-chain nor PDGF ß-receptor mRNAs were detected in the injured vessels 2 weeks after injury once cell proliferation had stopped. PDGF {alpha}-receptor mRNA was not detected in the injured arteries at any time and served as a useful negative control for these hybridizations. Additional slides hybridized with 35S-labeled PDGF A-chain or PDGF ß-receptor riboprobes transcribed in the sense orientation were also negative, confirming the specificity of the hybridization reaction.

The finding of increased cell proliferation and growth factor synthesis in the adventitia relative to the medial wall at early times after angioplasty suggested that the adventitia may play a role in the formation of the subsequent vascular lesion. To determine the fate of the proliferating adventitial cells, we sought to label this cell population at an early time with BrDU and then examine the distribution of these cells once the neointima was fully formed. Animals were injured on day 0 and injected with BrDU between days 2 and 3, and the distribution of BrDU-positive cells was examined on day 14 without subsequent BrDU injection. Since the adventitial cells constitute the major population of proliferating cells on day 3, we expected that this would enable us to determine whether these cells had migrated from their position in the adventitia and contributed to the cellular mass in the neointima. This approach had been used previously to follow the migration and subsequent proliferation of medial cells into the neointima in the rat carotid injury model by [3H]thymidine labeling.28 Two weeks after angioplasty, a large number of cells labeled with BrDU between days 2 and 3 were still found in the adventitia surrounding the injured vessel; however, 43.1±3.3% (mean±SEM) of the neointimal cells were BrDU positive as well (Fig 4Down). The distribution of labeled cells tended to form a continuum extending from the adventitia into the neointima. The majority of these cells were very lightly stained, suggesting that they may have undergone subsequent replication with loss of labeled DNA to their daughter cells since incorporating the BrDU label between days 2 and 3. These data suggest that some of the proliferating adventitial cells may have migrated from the adventitia across the remaining external elastic lamina and contributed to the cellular mass in the neointima.



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  		Figure 4. Identification of proliferating cells by double-label immunohistochemistry using antibodies directed against BrDU (blue), {alpha}-smooth muscle actin (brown) (A and B), smooth muscle myosin (brown, C), or h-caldesmon (brown, D) in animals that received BrDU between days 2 and 3 after angioplasty and were killed on day 3 (A) or day 14 (B through D) without subsequent BrDU administration. Administration of BrDU between days 2 and 3 predominantly labels adventitial cells when analyzed on day 3 (A and TableUp). Double-label immunohistochemistry on these tissues indicated that these cells do not contain {alpha}-smooth muscle actin (A), smooth muscle myosin, or h-caldesmon (data not shown). To determine the fate of the cells proliferating on day 3, animals were injected with BrDU between days 2 and 3 and were killed on day 14 without subsequent BrDU administration (n=2). Quantification of the number of BrDU-labeled cells in hematoxylin-counterstained sections indicated that 43.1±3.3% (mean±SEM) of the neointimal cells had taken up the BrDU label between days 2 and 3. In addition, a number of cells that had proliferated between days 2 and 3 were found remaining in the adventitia on day 14. Double-label immunohistochemistry indicated that the BrDU-positive cells stained uniformly with {alpha}-smooth muscle actin antibodies in both the adventitia and neointima (B). Comparison of the adventitial BrDU/{alpha}-smooth muscle actin staining in A and B suggests that the adventitial cells changed phenotype and increased production of {alpha}-actin by day 14. Smooth muscle myosin was colocalized with the BrDU-positive cells in the intima and some of the adventitial cells (C). h-Caldesmon staining was not uniform throughout the neointima, and only a few BrDU-positive cells in that region also stained with h-caldesmon (D). h-Caldesmon staining was much more specific for the medial SMCs and was absent in the adventitia. The localization of smooth muscle actin and myosin in the intima and adventitia and the corresponding lack of staining with h-caldesmon in the adventitia and intima is consistent with our identification of the proliferating adventitial cells and possibly some of the intimal cells as myofibroblasts. Arrows indicate the border of the broken end of the media. Sections shown in A and B were stained in parallel for an extended period in the alkaline phosphatase substrate solution to emphasize the staining of the cells in the neointima in B. Magnification x32.

Immunohistochemistry was performed to identify the proliferating adventitial cells as well as the cells that migrated to form the neointima. Single-label immunohistochemistry using antibodies against {alpha}-smooth muscle actin, smooth muscle myosin, h-caldesmon, or desmin consistently labeled SMCs in the medial wall of normal arteries but did not stain adventitial cells, with the exception of a few SMCs surrounding small adventitial vessels. Consistent with previous work, vimentin staining of normal vessels was distributed throughout the adventitia and in the medial SMCs.25 Single- or double-label immunohistochemistry with {alpha}-smooth muscle actin, smooth muscle myosin, h-caldesmon, desmin, or vimentin and the BrDU antibody on tissues from animals injected with BrDU between days 2 and 3 and killed on day 3 revealed a very similar pattern of staining and indicated that the proliferating adventitial cells contained vimentin but were negative for all of the smooth muscle–specific markers (Fig 4AUp).

A very different pattern of antibody staining in the adventitia was seen 2 weeks after PTCA. Smooth muscle {alpha}-actin staining was found to extend into the surrounding adventitial space well beyond the medial wall, including regions in which the proliferating cells had been found on day 3. Double-label immunohistochemistry with the {alpha}-actin and BrDU antibodies on vessels from animals that received BrDU between days 2 and 3 and were killed on day 14 indicated that the previously actin-negative adventitial cells that had proliferated on day 3 now showed strong {alpha}-actin staining (Fig 4AUp versus 4B). A similar distribution of staining was seen with vimentin, except that vimentin staining also extended into regions of the adventitia that did not proliferate on day 3 and extended well beyond the actin-positive regions (data not shown). Strong vimentin and {alpha}-actin staining was also found in the neointima and in the medial wall. In the neointima, almost all of the BrDU-positive cells showed smooth muscle {alpha}-actin and vimentin staining. Staining with the other SMC markers was much more limited and did not include the adventitial cells, which had proliferated on day 3: smooth muscle myosin was found in the media and neointima and weakly stained some cells in the adventitia (Figs 4Up and 5Down); h-caldesmon staining was restricted to the medial wall with some scattered positive intimal cells but did not stain the adventitial cells (Figs 4Up and 5Down); and desmin staining was found in the medial SMCs alone (data not shown). Segments of the coronary arteries proximal or distal to the injury site did not show similar changes in adventitial {alpha}-actin staining (Fig 5ADown versus 5D). Together, these data suggest a phenotypic switch of the adventitial cells, which are responding to balloon injury by increasing {alpha}-actin content.



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  		Figure 5. Double-label immunohistochemistry 14 days after angioplasty using antibodies directed against BrDU (blue) and smooth muscle markers (brown) of the left anterior descending coronary artery from an animal that received BrDU between days 2 and 3. Double-label immunohistochemistry was performed as described in Fig 3Up using SM1 (A and D), smooth muscle myosin (B; 1/1000 dilution, Sigma), and h-caldesmon (C; 1/1000 dilution, Sigma) antibodies. Comparison of the injury site (A through C) with the proximal uninjured vessel from the same animal (D) suggests that the increase in adventitial actin-positive cells occurs in the adventitia specifically at the site of injury and includes most if not all of the cells that had proliferated in the adventitia between days 2 and 3. The intimal cells stained with SM1 and smooth muscle myosin, but only a few cells contained h-caldesmon. The adventitial cells reacted strongly with the {alpha}-smooth muscle actin antibody SM1 but only weakly with the smooth muscle myosin antibody and not at all with the h-caldesmon antibody. Magnification: A through D, x8.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present series of experiments, we have characterized cell proliferation and growth factor expression after balloon overstretch injury of porcine coronary arteries using standard PTCA balloon catheters. Although the time course of cell proliferation was not appreciably different from that seen previously in rats and rabbits in which denudation-type balloon angioplasty was examined in peripheral vessels,7 29 cell proliferation at the earliest time point after angioplasty of porcine coronary arteries was greatest in the adventitia compared with the media. These data suggest that the adventitia may also be an important region with respect to the first wave of growth after angioplasty of coronary arteries.

We hypothesize that the proliferating adventitial cells migrate into the neointima. An examination of BrDU labeling in the neointima of animals killed on day 14 indicated that 43% of the neointimal cells arose from cells that had incorporated BrDU between days 2 and 3. Since the largest proportion of cells incorporating BrDU at that time is found in the adventitia, this suggests that some of the neointimal cells must be the daughter cells derived from the adventitial cells that incorporated BrDU between days 2 and 3 and migrated into the lesion. This is essentially the same experimental evidence that was used to prove that neointimal cells in the rat carotid injury model arise from the medial wall.28 Additional work will have to be done to establish the exact percentage of cells migrating from the adventitia into the neointima.

We hypothesize that the cells that proliferate in the adventitia may also contribute to vascular lesion formation by synthesizing growth and/or differentiation factors. These cells synthesize PDGF A-chain and PDGF ß-type receptor mRNAs and may therefore affect growth or differentiation of the proliferating cells in the adventitia, media, or intima. In vitro studies indicate that PDGF (consisting of both A and B chains) is a mitogen43 and chemoattractant44 for SMCs and fibroblasts. However, more recent studies have questioned the role of PDGF B chain as a growth factor in vivo.45 Additional work suggests that PDGF A chain and PDGF B chain may have different effects on smooth muscle growth and differentiation.46 47 48 However PDGF is acting, there is a colocalization of PDGF A-chain and/or PDGF ß-receptor mRNAs in proliferating cells after vascular injury in rats,49 primates,50 51 human lesions,52 and now pigs, from which one may infer a role for this factor in lesion formation. PDGF A-chain and B-chain and ß-receptor mRNAs are found in human atherosclerotic plaques by in situ hybridization,53 localized predominantly in mesenchymal-appearing intimal cells. The mesenchymal-appearing intimal cells are stellate, display variable amounts of cytoplasm, and have large pale hematoxylin-staining nuclei but do not stain well with antibodies directed against {alpha}-smooth muscle actin or markers for endothelial cells, macrophages, or T cells. SMCs may show variable {alpha}-smooth muscle actin staining and will often take on a "mesenchymal" or "fibroblast-like" morphology when they proliferate both in vivo and in vitro.47 54 55 56 We have previously hypothesized that the mesenchymal-appearing intimal cells were derived from SMCs in the atherosclerotic plaque or media.53 Given the finding that adventitial myofibroblasts may participate in vascular lesion formation, we must consider the possibility that some of the mesenchymal-appearing intimal cells in atherosclerotic plaques may be derived from the adventitia or fibroblasts instead of vascular SMCs.

Balloon overstretch injury of porcine coronary arteries tears the media wall and exposes the outer elastic lamina.16 18 19 Vascular lesion formation then occurs in the region between the broken ends of the media on the luminal side of the internal elastic lamina from SMCs, which have been thought to arise from the broken ends of the medial wall. Similar tearing of the intima and media occurs as a result of clinical PTCA and is the primary mechanism for luminal enlargement by angioplasty.2 Another porcine coronary angioplasty model has been described in which tantalum stents are implanted by use of oversized balloon catheters.16 This model creates deep medial injury and stimulates thrombus formation, which is then organized into a neointima that resembles the postangioplasty restenosis lesion. Lesion formation in this model has been suggested to be driven primarily from thrombus organization, and the neointimal lesion forms from the lumen in toward the media rather than from the adventitia toward the lumen. The neointimal SMCs after placement of tantalum stents are thought to arise from the adjacent normal medial wall.57 58 This is in direct contrast to the overstretch injury model, in which there is very little thrombus formation and few inflammatory cells18 19 and which appears to form from the adventitia toward the lumen. A major difference between the stent model and our present work is the use of tantalum stents to create deep medial injury leading to thrombus formation and lesion development.17 It has been suggested that the tantalum wires create a foreign body reaction, stimulating a local inflammatory response that is not seen after balloon injury alone.18 Minimal thrombus formation was observed in the present series of experiments.

The role of the adventitia in vascular lesion formation has been largely ignored despite numerous studies that have suggested its potential importance. Stripping of the adventitia stimulates vascular lesion formation and has been used as a model for atherosclerosis or postangioplasty restenosis research.59 60 61 In addition, changes in the expression of genes such as tissue factor62 or angiotensinogen63 have been observed in the adventitia after balloon catheter injury of rat aortas. Administration of drugs via the adventitia can stimulate vascular lesion formation. One of the first demonstrations of this was the placement of an endotoxin-treated thread in the adventitia outside the rat aorta.64 The thread stimulated a local inflammatory response in the adventitia, eventually resulting in the generation of a vascular lesion in the adjacent vessel. Adventitial drug delivery has been demonstrated to effectively deliver agents to the medial wall,65 which may be used as a strategy to prevent vascular lesion formation after angioplasty. Antisense oligomers directed against c-myb,66 heparin,67 68 or calcium antagonists69 placed in the adventitia have all effectively inhibited vascular lesion formation after denudation-type balloon catheter injury of rat carotid arteries. It has been assumed that these drugs acted directly on the medial SMCs and that the adventitia was simply a convenient route for administration. However, on the basis of the present findings, it may be equally valid to hypothesize that these experiments worked through a direct action on adventitial cells rather than the medial SMCs.

These studies suggest that the adventitia may play a role in vascular lesion formation after balloon overstretch injury of pig coronary arteries by contributing to the cellular mass of the neointima and the synthesis of growth factors. In addition, the adventitia may contribute to vascular remodeling and constriction of the external elastic lamina through an accumulation of myofibroblasts containing {alpha}-smooth muscle actin in the adventitia surrounding the injury site. Clearly, additional work will have to be directed at a more detailed examination of the response of adventitial cells to balloon injury and the role that these cells might play in the formation of vascular lesions and luminal narrowing associated with postangioplasty restenosis.


*    Selected Abbreviations and Acronyms
 
BrDU = 5-bromo-2-deoxyuridine
h-caldesmon = high caldesmon
PDGF = platelet-derived growth factor
PTCA = percutaneous transluminal coronary angioplasty
SMC = smooth muscle cell


*    Acknowledgments
 
This work was supported by NIH grant HL-47838-03 (Dr Wilcox), the Robert Wood Johnson Foundation for Minority Faculty Development (Dr Scott), and the Andreas Gruentzig Cardiovascular Center.

Received April 25, 1995; revision received December 21, 1995; accepted January 2, 1996.


*    References
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