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Estrogen attenuates the adventitial contribution to neointima formation in injured rat carotid arteries

Suzanne Oparil, Shi-Juan Chen, Yiu-Fai Chen, Joan N Durand, Leslie Allen, John A Thompson
DOI: http://dx.doi.org/10.1016/S0008-6363(99)00240-0 608-614 First published online: 1 December 1999


Objective: This study tested, in ovariectomized rats, whether (1) adventitial activation plays a role in the vascular injury response, and (2) inhibition of adventitial activation and the subsequent wave of cell proliferation moving from adventitia to neointima contributes to the estrogen-induced attenuation of neointima formation in balloon injured carotid arteries. Methods: Ovariectomized Sprague-Dawley rats were treated with either 17β-estradiol or vehicle beginning 72 h prior to balloon injury of the right common carotid artery and were sacrificed at 0, 3, 7, 14 and 28 days after injury. BrdU was administered 18 h and 12 h prior to sacrifice in order to quantitate mitotic activity in adventitia, media and neointima of the damaged vessel at specified times post injury. Results: Adventitial activation, evidenced by positive BrdU staining, was evident on the day of injury, peaked on day 3 and was resolved by day 7, thus preceding neointima formation. Numbers of BrdU labeled cells in adventitia on day 3 were significantly reduced in estrogen treated rats compared to controls. BrdU labeled cells were undetectable in media on the day of injury, appeared at day 3 and disappeared by day 14. Neointima appeared at day 7 and increased in area throughout the period of observation. Neointimal area and numbers of BrdU labeled cells in neointima were significantly reduced in estrogen treated rats compared to controls. These findings suggest that there is a wave of cell proliferation moving in an adventitia-to-lumen direction following endoluminal injury of the rat carotid artery and that estrogen modulates this proliferative response to injury. Conclusion: These results support the hypothesis that adventitial activation contributes to the vascular injury response and that estrogen reduces this contribution.

  • Hormones
  • Gender
  • Restenosis
  • Smooth muscle
  • Arteries

Time for primary review 22 days.

1 Introduction

Estrogen inhibits the development of atherosclerotic cardiovascular disease in women [1]. Mechanisms that have been proposed to account for this vasoprotective effect are complex and include alterations in lipid metabolism, endothelial function, smooth muscle cell proliferation and associated extracellular matrix formation, as well as vascular reactivity. Previous studies in our laboratory have demonstrated a sexual dimorphism (female<male) in the neointimal response to balloon injury of the rat carotid artery and have shown that estrogen is responsible for the dimorphic injury response [2–6]. We have further shown that estrogen inhibits the response to vascular injury via mechanisms involving stimulation of re-endothelialization and recovery of endothelial cell (EC) function [6–8]. While the latter mechanisms are undoubtedly important in modulating the extent of the vascular injury response, they appear to come into play relatively late in the course of neointima formation after balloon injury. Neither functional nor structural re-endothelialization of damaged carotid arteries is impressive at two weeks post injury in the absence of estrogen supplementation and therefore appears not to account for the sexual dimorphism of the early (<2 weeks) injury response.

Neointima formation and/or atherosclerotic lesions have been observed in response to adventitial injury in various animal models [9–12], raising the possibility of alternative pathways of the injury response and alternative routes of administering therapeutic agents. More recently, endoluminal injury of porcine coronary artery was shown to result in significant remodeling of the adventitia, which included proliferation and differentiation of adventitial fibroblasts to myofibroblasts, characterized by expression of α-smooth muscle actin [13]. These adventitial responses were associated with increased neointima and extracellular matrix formation [13–15]. These findings suggest that adventitial fibroblasts may contribute to the response to endothelial/medial vascular injury. The role of this process in the rat carotid injury response and its modulation by estrogen were examined in the current study.

2 Methods

2.1 Animals

Ten-week old female S-D rats were obtained from Charles River Breeding Laboratories (Wilmington, MA). All rats were maintained at constant humidity (60±5%), temperature (24±1°C), and light cycle (6 AM to 6 PM) and were fed a standard rat pellet diet (Ralston Purina Diet) ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No 85-23, revised 1996).

2.2 Ovariectomy and hormone treatment

Ten week old rats were subjected to ovariectomy [2] and assigned at random to treatment with daily injections of estrogen (17β-estradiol 20 μg/kg/d in 100 μl cottonseed oil, s.c.) or vehicle (100 μl/d cottonseed oil, s.c.). The 17β-estradiol was purchased from Sigma Chemical Co. This dose of 17β-estradiol has been shown in previous studies to result in serum 17β-estradiol concentrations of 30–40 pg/ml, well within the normal range of serum estradiol levels for intact female rats reported in the literature (30–50 pg/ml, depending on the stage of the estrus cycle) and previously observed in our laboratory [3].

2.3 Balloon-injury procedure

After 3 days of hormone or vehicle treatment, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and the right carotid artery was isolated by a middle cervical incision, and suspended on ties. The distal right common carotid artery and region of the bifurcation were exposed. A 2F Fogarty balloon catheter (Baxter V. Mueller) was introduced through the external carotid artery and advanced into the thoracic aorta. The balloon was inflated with saline to distend the common carotid artery and was then pulled back to the external carotid artery. After six repetitions of the procedure, the endothelium was removed completely, as assessed by the Evans blue dye technique, and there was some injury to medial smooth muscle layers throughout the common carotid artery. After removal of the catheter, the external carotid artery was ligated and the wound closed. The left carotid artery was not damaged and served as a control. In a separate group of animals, the right common carotid artery was exposed and subjected to the same manipulation as the balloon injured vessel except for balloon insertion, thus serving as an additional sham surgical control.

2.4 Morphometric and in-situ analyses of injured vessels

In order to assess cellular proliferation and migration in balloon injured rat carotid arteries in the presence and absence of estrogen treatment, the bromodeoxyuridine (BrdU) technique was utilized [16,17]. BrdU is specific for labeling DNA in the S phase of the cell cycle. Ovariectomized rats (n=6 per treatment group and per time point) that were subjected to balloon injury of the carotid artery and treated with 17β-estradiol or vehicle were sacrificed at 1, 3, 7, 14, 21, and 28 days after injury. Sham surgical control animals were sacrificed at 3, 7 and 14 days after the procedure.

BrdU was administered 18 h (30 mg/kg, i.p.+100 mg/kg, s.c.) and 12 h (30 mg/kg, i.p.) prior to sacrifice for detection and quantitation of cells that had entered into the cell cycle within 24 h of sacrifice. Rats were killed with an overdose of sodium pentobarbital (75 mg/kg) at the time points specified above and perfused with 10% formalin at a pressure of 120 mm Hg. The vascular system was rinsed with phosphate-buffered saline (pH 7.4) before infusion of fixative solution. Both carotid arteries were isolated from adherent tissue and fixed in 10% formalin for 24 h prior to sectioning. Vessels were embedded in paraffin, and cross sections of vessels were cut every 3 mm and processed for immunohistochemistry using anti-BrdU antibodies (BrdU-Cell Proliferation Kit, Amersham). Sections were counter stained with hematoxylin/eosin. Three sections with good attachment of adventitia and neointima to media were selected from the middle third of the injured region of each vessel for measurement. The mean of the three measurements was used for statistical analysis. The proportion of BrdU-positive cells was determined by cell counts under light microscopy using a computer-based Image-Pro Morphometric system. The extent of proliferative activity was quantitated by determining the proportion of BrdU-positive cells in the adventitia, media and neointima of each cross section. All of the cells were counted by two independent observers, who were blinded with respect to the experimental group to which each sample belonged. The ratio of the positively stained cell population to total cells was calculated. The rat ileum served as a positive control for BrdU staining, and the undamaged left carotid artery and sham operated right carotid artery served as controls for vascular injury.

In all cases, morphometric analysis of damaged carotid arteries was also carried out to assess the extent of the vascular injury response. The cross-sectional surface areas of the vessel within the external elastic lamina (total area), within the internal elastic lamina (intimal area), and within the lumen (lumen area) were measured. The extent of neointima formation in the injured carotid artery was expressed as the absolute area of neointima and the I/M ratio.

2.5 Statistical analysis

Results are expressed as means±SEM for each group. Statistical analysis was carried out using the CRUNCH statistical package (CRUNCH Software Co., Oakland, CA) on an IBM 486 compatible computer. Initial statistical comparisons were performed with two-way or one-way analysis of variance. If analysis of variance results were significant, a post-hoc comparison among groups was performed with the Newman-Keuls test. Differences were reported as significant if the P value was <0.05.

3 Results

Both the damaged right and undamaged left carotid arteries were examined histologically after perfusion fixation at 1, 3, 7, 14, 21, and 28 days after injury. In the undamaged left carotid artery of balloon injured rats and in the right carotid artery of sham operated rats, the intima was a single cell layer thick; the internal elastic lamina was intact and the external elastic lamina was in contact with the adventitia in all rats examined. There were no differences in the total area or the medial area (wall thickness) of the undamaged left carotid artery or the sham operated right carotid artery among experimental groups. Collectively, these observations suggest that the anatomy of the intact carotid artery was not significantly altered systemically by either vehicle or hormone treatment and that the anatomy of the sham operated right carotid artery was not altered by manipulation of the vessel.

Neointima, consisting of circumferentially uniform, multiple layers of cells, appeared for the first time 7 days after balloon injury; neointima was undetectable in the 1 and 3 day specimens (Figs. 1 and 2). In estradiol-treated rats, the extent of neointima formation in the damaged carotid artery was less than in vehicle treated animals at all time points (Figs. 1 and 2). The differential between neointimal area in estrogen-treated vs. vehicle control rats was greatest at 7 days post injury and tended to diminish over time but remained statistically significant at 28 days. Morphometric analysis showed that the neointimal area and the I/M ratio were significantly less in estrogen-treated rats than in vehicle controls (Figs. 1 and 2). There was no significant change in the total area or the medial area of damaged carotid arteries compared to undamaged vessels in either experimental group (Figs. 1 and 2).

Fig. 2

Effects of administration of 17β-estradiol (E2, 20 μg/kg/d) on neointima formation in balloon-injured right common carotid artery of vehicle and E2-treated ovariectomized S-D rats. Results are mean±SEM. n, number of rats. *P<0.05 vs. respective control groups.

Fig. 1

Representative light micrographs of right common carotid arteries (change) from vehicle-treated and 17β-estradiol (E2)-treated ovariectomized S-D rats 1, 3, 7, 14, 21,and 28 days after balloon injury. BrdU was administered 18 h (30 mg/kg, i.p.+100 mg/kg, s.c.) and 12 h (30 mg/kg, i.p) prior to sacrifice. a, adventitia; m, media; n, neointima. Magnification X400.

Adventitial activation, as evidenced by positive BrdU staining, occurred early following vascular injury and preceded neointima formation (Figs. 1 and 3). BrdU labeled cells were detectable in the adventitia on the first day of injury, prior to the appearance of BrdU labeling in media in both experimental groups. Numbers of BrdU labeled cells in adventitia peaked at 3 days and were greatly reduced by 7 days post injury, by which time large numbers of BrdU labeled cells had appeared in neointima. In estradiol-treated rats, numbers of BrdU labeled cells in adventitia were significantly lower than in vehicle treated animals at 3 days post injury, the time of peak adventitial activation. Adventitial mitotic activity was minimal at 14 and 21 days after injury and undetectable at 28 days. Due to the nature of the rat carotid artery preparation, in which the adventitia is disrupted in the course of harvesting the vessel, adventitial area and therefore absolute adventitial cell numbers and numbers and density of BrdU labeled cells could not be determined with precision and compared in estrogen treated and control groups (see Study Limitations). Instead, numbers of BrdU labeled cells per field were counted in sections in which adventitia was relatively intact and uniform and well attached to media, providing a semiquantitative assessment of the extent of adventitial activation and a basis for comparison between treatment groups. There was no evidence of adventitial activation (no BrdU labeled adventitial cells) in the sham operated vessels or in the uninjured left carotid arteries.

Fig. 3

Time course of appearance of BrdU labeled cells in adventitia, media and neointima in cross sections of balloon-injured right common carotid artery of vehicle (top) and E2-treated (bottom) ovariectomized (OVX) S-D rats. Results are mean±SEM. n-number of rats.

Baseline cellularity of the media of injured vessels was comparable in the two treatment groups, and no BrdU labeled cells were detectable in the media in either group on the day of injury. Numbers of BrdU labeled cells in media peaked at 3 days and BrdU labeling persisted at 7 days, reaching minimal to undetectable levels by 14 days in both experimental groups (Fig. 1). Numbers and density of BrdU labeled cells in media were significantly reduced at 3 and 7 days post injury in estrogen-treated rats compared to vehicle controls. Total cell number in media tended to increase from day 3 to day 7 in both experimental groups, although the increase did not attain the level of statistical significance, then declined to baseline levels by 14–21 days post injury. No BrdU labeled cells were detected in the media of either uninjured left coronary arteries or sham operated right carotid arteries at any time point in the study.

The newly formed neointima, which was first detected at 7 days post balloon injury, contained large numbers of BrdU labeled cells. Numbers of BrdU labeled cells in neointima were greatly reduced at 7 days post injury in estrogen-treated rats compared to vehicle controls. Neointimal BrdU labeling decreased dramatically by 14 days and was nearly undetectable thereafter in both groups (Fig. 1). Total neointimal cell number increased steadily from 7 to 28 days post injury in both groups, but was significantly lower in the estrogen treated group compared to the vehicle control group at all time points examined. The density of BrdU labeled cells in neointima peaked at 19% in vehicle control rats at 7 days post injury and was significantly different from estrogen-treated rats at that time point. The finding of a higher percentage of BrdU labeled cells in the smaller neointima of estrogen treated animals may be attributed to reduced mitotic activity, and therefore greater retention and reduced dilution of label in cells, and/or to reduced extracellular matrix formation in this group (see Study Limitations). BrdU cell density decreased to a very small (<1.0) percentage by 14 days post injury in both groups. Neointima was undetectable in uninjured left coronary arteries and sham operated right carotid arteries at all time points in the study.

4 Discussion

This study tested the novel hypotheses that adventitial cells are activated and contribute to neointima formation in balloon-injured carotid arteries of ovariectomized rats, and that estrogen inhibits these processes, thus limiting the extent of the injury response. Our results demonstrated that proliferative activity, demonstrated by immunostaining for BrdU, increased in adventitia in the first 3 days post injury, prior to neointima formation in both experimental groups. This was followed by a wave of proliferative activity moving in an adventitia-to lumen direction. Adventitial cells had become quiescent by the time neointima appeared at 7 days past injury. Estrogen treatment was associated with both diminished adventitial activation, as indexed by reduced numbers of BrdU labeled cells in adventitia at 3 days post injury, and reduced numbers of proliferating cells in media and neointima at subsequent time points. These findings provide indirect evidence in support of a new cellular mechanism by which estrogen inhibits the vascular injury response.

Previous studies by Gabbiani et al. [18,19], as reviewed by Zaleweski and Shi [20], have demonstrated that wound healing is associated with rapid activation of fibroblasts, which subsequently proliferate, migrate and undergo differentiation into myofibroblasts. Similar cellular responses have been described in a variety of other pathological conditions associated with fibrogenesis and organ remodeling [20–23]. Accordingly, it has been hypothesized that fibroblast activation, proliferation, migration and differentiation into myofibroblasts is a general characteristic of tissue repair, and therefore may also occur in the blood vessel wall in response to injury [20]. Balloon injury of the coronary vasculature of the pig has been shown to initiate a series of events similar to that previously reported for wound healing. These include increased proliferation of activated (BrdU-labeled) adventitial fibroblasts during the first 7 days post injury [13]; focal enlargement of the adventitia, due both to hypercellularity and deposition of extracellular collagen [15]; translocation of BrdU-labeled cells through the dissected media into the neointima; and transformation into a myofibroblast phenotype at 7–8 days post injury [14].

The current study provided a first indication that adventitial fibroblasts may also play a role in the response to endothelial/medial vascular injury of the rat carotid artery and that estrogen may attenuate the injury response in this model, at least in part, by inhibiting the activation of adventitial cells and their contribution to neointima formation. We do not interpret our results as excluding the participation of other cell types in neointima formation following endoluminal vascular injury. Previous studies in the rat carotid injury model have emphasized the role of medial smooth muscle cells (SMC) in the vascular injury response. There is general agreement that in the rat carotid artery, the response to endoluminal injury begins with destruction of the endothelium and damage to medial SMCs [24,25] and the internal elastic laminae. Thrombosis, release of circulating clotting factors and penetration of these factors into the vessel wall also contribute to the early phase of the injury response. Release of basic fibroblast growth factor (FGF-2) from dying SMCs follows within 24 h [26,27] and appears to stimulate medial SMC proliferation and to play an important role in determining the final extent of neointimal proliferation. Our current findings and data from other laboratories provide evidence that adventitial activation also occurs within the first 24 h following injury [13–15]. We hypothesize that the signaling pathway responsible for adventitial activation involves release of cytokines/mitogens from damaged SMCs that act on adventitial cells and stimulate their participation in the injury response.

Indirect evidence from in vitro systems suggests that estrogen modulates the vascular injury response by inhibiting growth factor-induced proliferation and migration of vascular cells. Studies carried out in human female aortic SMCs in culture, which were shown by Western and Northern blot analyses to express estrogen receptors (ERs), demonstrated estrogen-induced inhibition of 3H-thymidine incorporation and migration in response to FGF-2 and platelet derived growth factor B (PDGF-B) [28]. These effects were blocked by the ER antagonist tamoxifen, and thus appeared to be ER mediated. Migration of SMCs isolated from the aorta of female Sprague-Dawley rats in response to peak doses of the chemoattractants PDGF, insulin-like growth factor-1 (IGF-1) and fibronectin has been shown to be attenuated by 17β-estradiol in a concentration-dependent manner [29]. This effect was inhibited by the antiestrogen ICI 164384 and by actinomycin D, suggesting that SMC migration is ER mediated and dependent on synthesis of new DNA.

More recently, our own laboratory has tested the hypothesis that adventitial cells from carotid artery of female rats can be activated and stimulated in vitro to migrate in response to conditioned media from SMCs and that this process is modulated by estrogen [30]. In these studies, primary adventitial fibroblasts were recovered from polytetrafluoroethylene (PTFE) sponges coated with collagen and treated with recombinant human acidic fibroblast growth factor (FGF-1) that had been implanted for 3 weeks in the neck cavity of intact female S-D rats adjacent to the carotid adventitia. Using a Boyden chamber assay system, we demonstrated migration of adventitial fibroblasts in vitro in response to conditioned media from rat aortic SMCs. Migration was inhibited by 17β-estradiol in a concentration-dependent (10−9−10−7 M) and estrogen receptor-dependent (blocked by ICI-182780) manner. These in vitro experiments suggest that VSMC produce chemoattractant factor(s) that function as migratory stimuli for adventitial fibroblasts. The production and/or action of these factors are inhibitable by estrogen. These findings support the novel hypothesis that damaged medial SMCs and their cellular products are involved in adventitial activation and in directing the migration of adventitial cells into neointima and that these processes are modulated by estrogen.

4.1 Study limitations

The demonstration of adventitial cell activation in the current study was based on the indirect method of labeling cells within the arterial wall with BrdU and observing altered patterns of BrdU labeling over time following injury. In this study, BrdU would be expected to label all cells that were dividing (contained DNA in the S phase of the cell cycle) in the hours immediately following BrdU injection, regardless of cell type or location. Thus, the method did not selectively identify cells of adventitial origin or fibroblasts. The absence of cellular markers specific for fibroblasts which distinguish them from smooth muscle cells and inflammatory cells is a limitation of this, as well as previous studies of the adventitial contribution to the vascular injury response [13,14]. Ongoing studies in our laboratory are attempting to circumvent this limitation by marking syngeneic fibroblasts that have been harvested from the adventitia of rat carotid arteries by transducing them with retroviral particles (pLBg) coordinating expression of lacZ [31]. Transduced fibroblasts can then be reintroduced into the adventitia of injured carotid arteries and their migratory and proliferative behavior observed. Preliminary observations using this technique suggest that transduced adventitial fibroblasts have the capacity to migrate into the neointima following vascular injury. These studies are preliminary, however, and the adhesion/migration properties of the transduced fibroblasts and their modulation by estrogen have not been fully characterized.

Limitations of the BrdU labeling technique as a method for assessing the migration of adventitial cells to neointima, as pointed out by Shi et al. [14], include: (1) inability to identify adventitial cells that enter the cell cycle prior to or following a 6 hour window following BrdU administration and subsequently migrate to the neointima; (2) failure to identify nonreplicating cells that migrate from adventitia to neointima; (3) decreasing intensity of BrdU staining with time, likely as a result of the dilutional effect of ongoing cell divisions, with attendant loss of the label from some cells, making it difficult to use this technique to follow labeled cells over prolonged periods. Accordingly, we did not attempt to assess adventitial cell migration directly in the current study.


This work was supported in part by grants HL-07457, HL-45990, and HL-57270 from the National Heart, Lung and Blood Institute and a Grant-in-Aid (9750665N) from the American Heart Association. The authors thank Mark Evces and Marla Kolarik for their assistance in the preparation of the manuscript.


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