Coronary artery apoptosis in experimental hypercholesterolemia

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Atherosclerosis 142 (1999) 317 – 325

Coronary artery apoptosis in experimental hypercholesterolemia David Hasdai a, Giuseppe Sangiorgi a, Luigi G. Spagnoli b, Robert D. Simari a, David R. Holmes Jr. a, Hyuck Moon Kwon a, Paula J. Carlson a, Robert S. Schwartz a, Amir Lerman a,* a

Di6ision of Internal Medicine and Cardio6ascular Diseases, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, USA b Cattedra di Anatomia ed Istologia Patologica, Department of Surgery, Uni6ersita’ di Roma-Tor Vergata, Vergata, Italy Received 26 March 1998; accepted 14 August 1998

Abstract The altered coronary vasoactivity detected in experimental hypercholesterolemia before lesion formation is presumably due to an imbalance between vasodilating and vasoconstricting factors. Apoptosis, which has been previously described in advanced atherosclerosis, is modulated by vascular derived peptides with vasoactive properties. We hypothesized that coronary apoptosis occurs in experimental hypercholesterolemia prior to lesion formation. Pigs were sacrificed after being on either a high-cholesterol diet for 10–16 weeks (n= 17) or a normal diet (n= 9). Identification of apoptosis in each layer of coronary arteries and arterioles was performed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL). In additional animals, ligation-mediated polymerase chain reaction (PCR) and transmission electron and confocal microscopy were done. Plasma cholesterol levels were higher in the cholesterol-fed animals (86 99 mg/dl versus 342920 mg/dl, P B 0.001). Atheromatous plaques were not evident in the high-cholesterol group. TUNEL was positive in 11 of 17 hypercholesterolemic animals, primarily in the intima (1–2% of cells) and adventitia (3% of cells), but not in control vessels. A similar distribution was detected in arterioles. DNA bands were detected only in experimental animals, as were morphological features of apoptosis by transmission electron and confocal microscopy. In experimental hypercholesterolemia, apoptosis occurred in coronary arteries and arterioles before lesion formation. Apoptosis may be an integral process of early coronary atherosclerosis. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Apoptosis; Coronary artery disease; Hypercholesterolemia; Atherosclerosis; Pig; Adventitia; Intima

1. Introduction Apoptosis is a mode of cell death in which single cells are deleted in the midst of living tissue [1]. In contrast to cell necrosis characterized by organelle and cytoplasmic swelling culminating in cell disintegration, apoptosis produces compaction and segregation of chromatin at the periphery of the nucleus and condensation of the cytoplasm, followed by nuclear fragmentation and the budding off of intact cell fragments which may be phagocytosed by neighboring cells or macrophages [2]. * Corresponding author. Tel.: +1-507-2554152; fax: + 1-5072552550; e-mail: [email protected]

Apoptosis accounts for most or all of the programmed death responsible for tissue remodeling and atrophy, and often for the physiological death of cells in the course of normal tissue turnover [1]. Arterial remodeling also occurs in pathophysiological states, such as coronary artery atherosclerosis [3–5]. Accordingly, apoptosis has been detected in arteries with advanced atherosclerosis and after mechanical trauma [6–13]. However, it has not yet been determined whether apoptosis is an end-result of atherosclerosis or is an integral process of early atherogenesis. Prolonged diet-induced hypercholesterolemia in the pig causes rapid cellular turnover, eventually culminating in the formation of the atherosclerotic plaque [14–

0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 8 ) 0 0 2 4 9 - 4

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16]. We have recently reported that after only 10 weeks of high-cholesterol diet in the pig, coronary vascular reactivity is altered despite the absence of gross histopathologic changes associated with atherosclerosis [17– 21]. This altered vasoreactivity extends from epicardial conductance arteries to resistance vessels. Moreover, we have demonstrated that experimental hypercholesterolemia causes a shift in the balance between endogenous vasoconstrictor and vasodilator factors [19]. Pollman et al. [22] recently established that endogenous vasoactive agents such as angiotensin II and nitric oxide may also differentially modulate apoptosis. Thus, the shift in balance between vasoconstrictor and vasodilator factors resulting in altered coronary vasoreactivity in experimental hypercholesterolemia may be coupled with coronary artery apoptosis. This study was therefore designed to examine our hypothesis that coronary artery and arteriolar apoptosis occur during early atherogenesis in porcine experimental hyper-cholesterolemia, characterized primarily by functional rather than structural impairments in the integrity of the coronary vascular bed.

2. Materials and methods

2.1. Coronary artery specimens The following studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee. All experiments were conducted using juvenile domestic crossbred pigs weighing 25 – 37 kg. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985). The animals were placed on either a normal diet or a high-cholesterol diet of 2% cholesterol and 15% lard by weight (TD 93296, Harlan Tekiad) for 10–16 weeks. The animals were then euthanized using an intravenous commercial euthanasia solution by ear vein (Sleepaway, Fort Dodge Laboratories). Sections were cut transversely at random from the proximal segments of coronary arteries and from arterioles (B 400 mm in diameter). Standard hematoxylin-eosin and elastic van Gieson stains of epicardial sections were prepared. Quantitative histomorphometric measurements were obtained using a calibrated microscope, video-imaging system, and microcomputer program (JAVA, Jandel Scientific, Corte Madera, CA). Calibration was done prior to each measurement session. Arterial sections stained with elastic van Gieson were manually traced with the following parameters measured: lumen area; area within the internal elastic lamina; area within the external elastic lamina; and area within the adventitia (restricted to the area extending to no greater than two times the medial thickness). Intimal area was calculated

by subtracting lumen area from the area within the internal elastic lamina. Medial area was calculated as the area between the internal and external elastic lamina. The adventitial area was calculated by subtracting the area within the external elastic lamina from the area within the adventitia. The intimal area/medial area ratio was then calculated.

2.2. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay Identification of apoptosis in coronary cross-sections was performed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique [23], using the Boehringer Manhein In-situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN) with a modified protocol. All slides were reviewed by at least two investigators who were blinded to the diet given. Apoptotic nuclei were identified by the presence of dark blue staining. The number of positively-stained cells in each layer of epicardial arteries was counted using ×40 magnification. In case of doubt concerning the staining of a specific cell, a third investigator was consulted and a consensus was reached. Cell counts in the adventitia were restricted to the area extending to two times the medial thickness, as determined by the aforementioned morphometric analysis. Results are presented as the number of cells per mm2 (the area of each layer as determined by morphometric analysis) as well as the percentage of cells relative to the total number of nuclei. Human prostate tissue, known to exhibit programmed cell death [24], was used as positive control. The prostate tissue was treated as described above, with exposure to DNAse I (Boehringer Mannheim, Indianapolis, IN; 0.01 mg/ml) for 10 min at room temperature prior to application of the TUNEL mixture. In addition, several specimens were exposed to all the reagents including swine serum without the addition of the Tdt enzyme. These slides served as negative controls for nonspecific staining. For quality control assurance, seven specimens were stained more than once. In all cases, there was agreement between the different slides obtained from the same specimen. In addition, in five animals, specimens were obtained and analyzed from different epicardial arteries, and in all cases there was agreement between the different specimens obtained from the same animal.

2.3. Confocal microscopy— propidium iodide assay As an additional assay for apoptosis in hypercholesterolemic pigs, we used a fluorescent DNA-binding dye, propidium iodide (PI), to evaluate nuclear chromatin morphology in additional control and hypercholesterolemic animals. Apoptosis has been defined by a

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number of ultrastructural criteria, including cell size and gross structure, membrane permeability and integrity, as well as chromatin and DNA structure [25– 27]. Immediately after removal from the heart, the proximal portion of the three main coronary arteries was cut into rings (3 – 5 per artery) 1 mm in thickness. Each ring was subsequently placed in 3 ml of DMEH21 (Gibco BRL, Gaithesburg, MD) culture media containing 20% fetal bovine serum (FBS, Gibco BRL) with penicillin (100 U/ml), streptomycin (50 mg/ml) and fungizone (25 mg/ml) and incubated in humidified atmosphere containing 8% CO2 at 37°C. After equilibration to this temperature, the tissue sections were labeled with PI (5 mg/ml; molecular probes) for 30 min in order to determine the morphological characteristics of apoptotic cells. In preliminary experiments incubations period ranging from 10 to 40 min with PI were evaluated; an incubation period of 30 min allowed the best detection of increased PI staining. After the incubation, coronary rings were washed three times in phosphate buffered saline for 5 min, cut longitudinally, and placed under a confocal microscope on a glass slide. This form of confocal microscopy is based on the optical sectioning abilities of the confocal microscope to obtain en face, sequential images of cells at depth up to 1 mm. In our experiments, both the intimal and adventitial part of the coronary rings were examined under the microscope. Condensed and/or fragmented apoptotic nuclei were identified by the presence of bright yellow/orange fluorescence. Experiments were performed in duplicate and analyzed on a confocal laser scanning microscope LSM 310 (Carl Zeiss, Germany) equipped with one argon/kripton laser. PI excitation took place with the laser tuned on an excitation wave length of 488 nm, and emission was collected trough 630/22 and 530/30 nm band-pass filters, respectively. Images were acquired with a 4 ×0 neofluor lens with 1.3 n.a., digitized with a matrix of 512×512 pixels with a resolution of 0.3125 mm/pixel and analyzed by using the LSM 310 software on a IBM work station. Control experiments were performed in the same conditions. The results were analyzed independently by two blinded pathologists with an intraobserver variability of B 10%.

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2.5. Ligation-mediated polymerase chain reaction To verify the occurrence of apoptotic cell death in the hypercholesterolemic animals, we analyzed genomic DNA isolated from the coronary arteries of additional control (n= 5) and hypercholesterolemic pigs (n= 5 after 10 weeks of high-cholesterol diet) according to the method of Staley et al. ([28]). PCR products (15 ml) were analyzed by electrophoresis through 1.5% agarose gels equilibrated in 45 mM Tris–borate, 1 mM EDTA buffer pH 8.0. After electrophoresis for 2 h at 6 V/cm in a mini-gel apparatus (CBS Scientific, Del Mar, CA), gels were stained by ethidium bromide (Pharmacia Biotech, Cambridge, UK) and photographed with a Polaroid MP4 camera on a UV transilluminator Bands ranging from 150 to 1000 bp were considered PCR products of apoptotic DNA. All PCR experiences were repeated in duplicates.

2.6. PCNA In seven control and hypercholesterolemic animals with positive TUNEL staining, cell proliferation was assessed using an assay for PCNA on paraffin-embedded coronary artery samples (Vector Laboratories). The number of cells in each layer of the vessel wall with positive staining was determined.

2.7. Data analysis Results are presented as mean9S.E.M. Differences between groups were analyzed using the unpaired Student’s t-test. All tests were two-tailed, and P values of 50.05 were considered significant.

3. Results In the preliminary experiments, 17 pigs were fed a high-cholesterol diet and nine pigs a normal diet. Plasma cholesterol levels were significantly higher in the cholesterol-fed animals, primarily due to higher lowdensity lipoprotein levels, but high-density lipoprotein levels were elevated as well (Table 1).

2.4. Transmission electron microscopy

3.1. Histology and morphometry

To provide morphological evidence of apoptosis, we performed transmission electron microscopy of epicardial arteries. Portion of coronary arteries from additional control and hypercholesterolemic pigs were fixed in 2.5% glutaraldehde (pH 7.3). Semithin sections were stained with toluidine blue and ultrathin section of the areas of interest stained with uranyl acetate and lead citrate and examined with a Phillips CM10 electron microscope.

The intimal area of epicardial sections was not significantly greater in hypercholesterolemic pigs (Table 1, P= 0.13), nor was the intima to media area ratio (P= 0.07). There were no significant differences in medial or adventitial area. In several animals in the high-cholesterol group myointimal thickening with scant accumulation of basophilic material and foam cells were observed using hematoxylin-eosin staining (Fig. 1). Although the in-

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Table 1 Lipid profile, morphometric analysis and density count of TUNEL- and PCNA-positive cells in the two study groups

Cholesterol (mg/dl) Low-density lipoprotein (mg/dl) High-density lipoprotein (mg/dl) Intimal area (mm2) Medial area (mm2) Adventitial area (mm2) Intima/media area ratio Positi6e TUNEL staining (cells/mm 2) a Intima Media Adventitia Positi6e PCNA staining (cells/mm 2) b Intima Media Adventitia a b

Normal diet

High-cholesterol diet

P value

869 9 529 10 43 9 9 0.06290.060 1.161 90.089 1.158 90.201 0.049 9 0.010

342 920 254 917 82 94 0.153 90.287 0.917 9 0.246 1.244 9 0.426 0.213 90.150

B0.001 B0.001 B0.001 0.13 0.24 0.18 0.07

– – – 13 910 1 91 139 4

173 958 591 1079 51

– – –

160 961 8 97 89 914

0.04 0.22 0.04

Mean cell count in animals with positive staining. Mean cell count in control (n= 7) and hypercholesterolemic pigs (n = 7).

tima was slightly thickened in several hypercholesterolemic pigs, atheromatous plaques encroaching on the vessel lumen were not detected. Sparse cells with condensed chromatin within the nuclear membrane, characteristic of apoptosis [29 – 31], were also visualized in the intima and adventitial layers of coronary arteries harvested from hypercholesterolemic animals (Fig. 1).

In hypercholesterolemic animals, PCNA-positive cells were also primarily detected in the intima and the adventitia (Table 1). In similarity to the proportion of TUNEL-positive cells, 2% of cells in the intima and 3% of cells in the adventitia were PCNA-positive.

3.2. In situ detection of apoptotic cells

Coronary artery rings from hypercholesterolemic animals were positively-stained with PI. Morphological assessment of the PI-positive cells revealed that almost of all were apoptotic, exhibiting cell shrinkage and nuclear rounding (Fig. 3). Alterations consistent with necrotic cell death, as indicated by nuclear and cytoplasmic swelling, were rarely observed. These results are in accord with previous studies using PI to detect apoptotic cells [7,32,33].

TUNEL staining for apoptosis was positive in 11 of the 17 hypercholesterolemic animals (65%), with variability in the number of cells stained for apoptosis between different specimens. In contrast, TUNEL stain was not detected in any of the control coronary arteries (Table 1), suggesting that this process occurs at a very limited rate or below detection in normal arteries. In the 11 hypercholesterolemic animals positively stained for apoptosis, apoptotic cells were primarily detected in the intima and adventitia, with few cells in the tunica media and the endothelial layer (Table 1 and Fig. 2a). In the 11 specimens with positive staining, 1 – 2% of cells in the intima and :3% in the adventitia were TUNEL-positive. Most of the TUNEL-positive cells demonstrated hyperchromatic and fragmented nuclei, which are characteristic morphological features of apoptosis. Other nuclei which were positively stained by the TUNEL technique were histologically intact, and thus they may represent cells in early apoptotic phases. Apoptosis was also detected in arterioles of hypercholesterolemic pigs (Fig. 2b). Specimens that were exposed to all the reagents including swine serum without the addition of the Tdt enzyme were negative in all cases.

3.3. Confocal microscopy and propidium iodide assay

3.4. Transmission electron microscopy By transmission electron microscopy, the coronary artery myointimal thickening observed in hypercholesterolemic pigs was mostly composed of smooth muscle cells and a lower number of macrophages both embedded in a matrix composed of ground substance, collagen and elastin. Scant monocytes and lymphocytes were evident. Cells with features consistent with apoptosis were detected in hypercholesterolemic, but not control pigs (Fig. 4).

3.5. Ligation-mediated polymerase chain reaction DNA samples obtained from hypercholesterolemic pig coronary arteries contained oligonucleosomal DNA strand breaks, whereas DNA strand breaks were not

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Fig. 1. Coronary artery specimen obtained from hypercholesterolemic pig and stained with hematoxylin-eosin ( ×50). Myointimal thickening is evident with accumulation of basophilic material (black arrows) and sparse foam cells (white arrows). The remaining population is composed by smooth muscle cells. Using high-power ( ×312.5) in another section, cells within the neointima with morphological features suggestive of apoptosis (condensation of chromatin around the nuclear membrane) were visualized (insert). Fig. 2. (a) Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) technique of a coronary artery obtained from a hypercholesterolemic animal ( ×50). Staining is positive in all layers of the vessel. In the adventitia, cells with positive staining (black arrows) were particularly present in the region corresponding to the external elastic membrane (as depicted using elastic van-Gieson staining). Several endothelial cells were also positively stained, as indicated by the arrow. Using high-power ( ×312.5) in another section, TUNEL-positive cells in the neointima exhibited nuclear chromatin condensation with peripheral addensation (insert), characteristic morphological features of apoptosis. (b) A similar pattern of staining was evident in coronary arterioles of hypercholesterolemic pigs ( ×50). Fig. 3. Confocal microscopy using propidium iodide (PI) in a coronary artery obtained from a hypercholesterolemic animal ( × 1280). In the tunica adventitia there are some nuclei with clumps of condensed chromatin, suggestive of apoptosis (open arrow). In the same field, residual apoptotic bodies, appearing as small clumps of condensed chromatin, are evident (filled arrow).

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Fig. 4. Transmission electron micrographs of sections of coronary arteries from normal and high-cholesterol treated animals. Note the normal ultrastructural aspects of a smooth muscle cell (panel A, × 12 000) in the media layer. Note also the presence of diffuse heterochromatin in the nucleus (panel B, × 32 000). In contrast, cells from hypercholesterolemic animals show varying degree of nuclear ultrastructural changes of apoptosis. In one cell there is lobulation of the nucleus (panel C, × 12 000) associated with slight compaction and margination of nuclear chromatin along the inner surface of the nuclear envelope (panel D, × 32 000) indicating an early stage of apoptosis. Another cell (panel E, ×9000) displays a more advanced apoptotic state with marked chromatin condenstation and peripheral margination. An apoptotic residual body is shown in panel F ( ×8000).

detected in control animals (Fig. 5). However, owing to the relatively low percentage of cells undergoing apoptosis as discerned by TUNEL, a smear like pattern was obtained rather than a clear ladder. This pattern has been previously described in human atherosclerotic plaques with a low percentage of apoptotic cells [34].

4. Discussion The principal finding of the present study is that there was biochemical and morphological evidence for coronary artery apoptosis in porcine experimental hypercholesterolemia. Although TUNEL staining was detected in all layers of the coronary artery wall, including the endothelial layer, it was predominantly evident in the intima and the adventitia of both epicar-

dial and arteriolar sections. Apoptosis was confirmed biochemically by the presence of DNA strands in highcholesterol pigs, but not control pigs. Morphologic features of various stages of apoptosis were detected by transmission electron and confocal microscopy in the coronary arteries of hypercholesterolemic pigs. These findings suggest that apoptosis may be an integral process in the changes occurring during early coronary atherosclerosis.

4.1. Pre6ious studies Several groups have recently reported evidence of apoptosis in a proportion of coronary specimens retrieved from patients with advanced coronary atherosclerosis [6,7,9,11,13]. Apoptosis was most prominent in macrophage-enriched areas, and pheno-

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typically altered smooth muscle cells and macrophages made up the bulk of the apoptotic cells [7]. Han et al. [7] have also shown that after injury of the rat iliac artery with a balloon catheter, cells in the neointima underwent apoptosis. Kockx et al. [12] demonstrated that in a rabbit model of diet-induced hypercholesterolemia (0.3% cholesterol for 16 – 27 weeks) there was a low rate of apoptotic cell death. Apoptosis was assessed within the plaque, not the vessel wall [12]. Although apoptosis has been suggested by these previous studies to be important in atherogenesis, it is not clear whether apoptosis is an integral aspect of the pathogenesis of early atherosclerosis or rather an end result of atherosclerosis.

5. Apoptosis and early atherosclerosis As mentioned above, apoptotic cell death has been previously detected in a rabbit model of hypercholesterolemia, characterized by the formation of plaques [12]. Our findings of apoptosis in pigs with diet-induced hypercholesterolemia extend this prior study [12], indicating for the first time that apoptosis occurs in the early stages of coronary atherosclerosis, characterized by altered coronary epicardial and arteriolar vasoactivity, but prior to the development of the obstructive atheroma [17–21]. Our current data demonstrate that apoptosis occurs in both coronary epicardial vessels and arterioles. These findings indicate that the altered

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coronary vasoreactivity may be coupled with cellular apoptosis, suggesting that two processes are governed by similar mechanisms. Apoptosis may be induced during early atherosclerosis through several pathways. It has been shown in vitro that oxidized lipoproteins may induce apoptosis of macrophages, smooth muscle cells, and endothelial cells [35–37]. Therefore, apoptosis in our model may be a direct response to increased lipids or their derivatives. Altered expression or accumulation of mitogenic growth factors and cytokines, whose levels may be altered in atherosclerosis, may also regulate the rate of apoptosis [10,38]. Indeed, altered levels of interleukin1b [39] and basic fibroblast growth factor [40] have been reported in patients with early atherosclerosis. Both factors may affect the induction and suppression of apoptosis [11,41]. Although prior studies have focused on the effect of mitogenic factors on apoptosis, recent studies have expanded this paradigm to vasoactive factors. An imbalance between vasoactive agents is postulated to cause endothelial dysfunction in early atherosclerosis [42]. It is therefore of interest that vasoactive agents such as angiotensin II and nitric oxide may also suppress or induce apoptosis [22]. Vasodilators such as nitric oxide suppress cell proliferation and induce apoptosis, whereas vasoconstrictors such as angiotensin II induce cell proliferation and suppress apoptosis. One may therefore speculate that apoptosis and the functional changes associated with early atherosclerosis are governed by common mechanisms. Indeed, agents such as angiotensin converting enzyme inhibitors, which inhibit the angiotensin pathway and augment nitric oxide activity, may induce apoptosis [43]. Concurrently, angiotensin converting enzyme inhibitors exert a salutary effect on coronary endothelial dysfunction [44].

5.1. Apoptosis and cell proliferation

Fig. 5. Ligation-mediated PCR of coronary arteries from hypercholesterolemic (lane 1a) and control (lane 2b) pigs. Hypercholesterolemic pigs demonstrated DNA strand breaks in mono- or oligo-nucleosomes, whereas DNA from control animals did not produce a clear laddering pattern. Lane 3c, molecular weight markers in bp.

In the present study, apoptosis was mainly detected in the intima (1–2% of cells) and adventitia layer (3%) of coronary arteries from hypercholesterolemic animals. This process was coupled with a marked increase in the number of proliferating cells in the coronary arteries of high-cholesterol treated animals compared to controls. This observation further supports the notion that apoptosis and proliferation occur in an inhomogeneous fashion, varying in time as well as in space, leading to variation in the number of cells between different regions within a tissue. One may hypothesize that in the earlier stage of a lesion development and at certain critical times in the evolution of a lesion, cell proliferation may predominate, while in later stages cell death may become dominant. In our model, after 10–16 weeks of cholesterol treatment, the apoptosis rate slightly exceeded indeed the proliferation rate. It is

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important to note that our data present a static view of advancing atherosclerotic lesions at a given point in time, not an integrated picture over the life of the lesion. Therefore, rather than establishing absolute quantitative rates of apoptosis and proliferation, these data should be used to support the concept that apoptosis occurs in the early phase of atherosclerotic lesion formation and should be added to our considerations of growth control and pathobiology of these lesions.

5.2. Ad6entitia and apoptosis Our data demonstrate evidence of cell proliferation and apoptosis in the adventitia of hypercholesterolemic pigs. The role of the adventitia in the development of coronary atherosclerosis continues to emerge. Several investigators have demonstrated that adventitial manipulation resulted in arterial intimal hyperplasia, which was exacerbated by a high-cholesterol diet [45 – 49]. In more recent studies, Scott et al. [50] reported that a large number of proliferating cells in the early stages after vascular injury were in the adventitia, whereas in later stages the proliferating cells were primarily in the neointima. The authors postulated that the proliferating adventitial cells may have migrated into the neointima. Shi et al. [51,52], extending these prior studies, showed that severe endoluminal coronary injury resulted in adventitial remodeling, and that the adventitial response was associated with neointimal formation. Given these findings, it is of interest that in our porcine model of diet-induced hypercholesterolemia apoptosis was primarily observed in the intimal and adventitial layers. One may speculate that programmed cell death accompanied the adventitial cell multiplication and migration to the neointima. It is possible that these processes occur in response to arterial wall hypoxia caused by cholesterol-induced damage to the vasa vasorum [53,54]. In addition, oxidized low-density lipoprotein may directly induce apoptosis [35 – 37] of adventitial and intimal cells.

5.3. Limitations In our study we did not detect evidence for apoptosis in control animals. Apoptosis occurs in physiological as well as pathophysiological states. However, the lack of detection of apoptosis in our control animals indicates that the rate of apoptosis in the steady-state is probably minimal and below the detection threshold of our techniques. In addition, the rate of apoptosis in hypercholesterolemic pigs was relatively low, corresponding to previously reported rates in hypercholesterolemic rabbits [12]. This may explain why TUNEL-positive cells were not detected in a proportion of hypercholesterolemic animals.

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