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Cardiovascular Research 1998 38(1):237-246; doi:10.1016/S0008-6363(97)00315-5
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Atherosclerosis in Marek's disease virus infected hypercholesterolemic roosters is reduced by HMGCoA reductase and ACE inhibitor therapy

Alexandra Lucasa,*,1, Erbin Daia,1, Li-Ying Liua,1 and Patric N Nationb

aDivision of Cardiology, John P. Robarts Research Institute, Box 5015, 100 Perth Drive, University of Western Ontario, London, Ontario, Canada, N6A-5K8
bDepartment of Lab Animal Services, University of Alberta, Edmonton, Alberta, Canada

* Corresponding author. Tel.: +1 (519) 6633214/4071; fax: +1 (519) 6633789; e-mail: arl@rri.uwo.ca

Received 14 May 1997; accepted 3 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Accelerated atherosclerosis is associated with herpesviral infection both in transplant patients and after balloon angioplasty. Marek's disease virus (MDV) is a herpesvirus that induces accelerated atherosclerosis associated with the development of an invasive lymphoma in hyperlipemic roosters. We have examined the effects of pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitor and quinapril, an angiotensin converting enzyme (ACE) inhibitor, on atherosclerosis development in MDV infected, cholesterol fed rooster chicks. Methods: The effects of these drugs on plaque growth after MDV infection were examined in two studies. In Study 1, MDV infected White Leghorn rooster chicks were divided into 4 groups assigned to normal or high cholesterol diet, and treated at three months of age with either pravastatin or saline. In Study 2, cholesterol fed rooster chicks infected with MDV were divided into 3 groups for treatment with either pravastatin, quinapril, or saline control. Results: A significant decrease in plaque area was detected after 60 days of treatment with both pravastatin and quinapril in cholesterol fed chicks (P<0.001). Lymphocyte infiltration into the arterial wall or target organs was not inhibited by treatment with either drug. Conclusions: (1) HMGCoA reductase inhibitor and ACE inhibitor therapy reduce atherosclerosis induced by virus infection and cholesterol diet, but this decrease in plaque growth is not due to a reduction in lymphocyte invasion. (2) MDV infection in cholesterol fed roosters provides a model for virus-induced arterial injury in atherogenesis.

KEYWORDS Marek's disease virus; Atherosclerosis; Angiotensin converting enzyme inhibitor; HMGCoA reductase inhibitor; White Leghorn roosters; Lymphocyte


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Rapid growth of atherosclerotic plaque occurs in two settings, after cardiac transplantation [1–3]and after angioplasty of stenotic coronary arteries [4, 5]. Viral infection has been implicated in plaque development in both settings, after transplant [6–9]and after balloon mediated arterial injury [10, 11]. Increased titers of antigenic markers for the herpesviruses, Cytomegalovirus (CMV) [9]and Herpes Simplex virus (HSV) [12], have been detected in smooth muscle cells at sites of de novo atherosclerotic plaque growth [13–16]. CMV infection has also been correlated with plaque development in transplant vasculopathy [6–9]. Viral antigens and DNA have been detected by immunohistochemical analysis [13, 14], in situ hybridization [13–15], and more recently by PCR analysis [15, 16]in atherosclerotic arterial extracts. CMV expression has also been associated with decreased expression of a tumor suppresser gene, p53, in areas of recurrent atherosclerotic plaque growth (restenosis) after angioplasty [10, 11].

Infection of newborn chicks with an avian gamma herpesvirus, Marek's Disease virus (MDV), induces both an invasive T cell lymphoma and atherosclerosis development in the thoracic aorta and larger arterial branches [17–19]. The precise mechanisms by which MDV and other herpesviruses initiate plaque growth, however, are not known. Damage induced by viral infection, altered cellular metabolism, direct cellular transformation [18, 20], and inflammatory and immune reactions to viral infection may accelerate plaque development [18–22]. MDV viral antigens have been detected in the arterial wall indicating viral infection of vascular cells and MDV has also been reported to increase smooth muscle cellular cholesterol levels in tissue culture [21]. MDV infection is associated with diffuse pervasive lymphocytic infiltrates which may increase vessel damage and initiate plaque growth. CMV infection of arterial smooth muscle cells has been detected in rats and is associated with increased mononuclear cell adhesion and invasion into the vascular endothelium [20, 22]. Cellular invasion in CMV infections is enhanced by high serum cholesterol levels [20, 22]. Monocyte and lymphocyte (specifically T cell) activation and invasion are associated with plaque development in animal models of balloon injury and hyperlipidemia [4, 23–25], but the role of lymphocyte invasion in herpesvirus, and specifically Marek's disease virus, associated atherosclerosis is not known.

The objective of this study was to assess the effects of the HMGCoA reductase inhibitor, pravastatin, and the ACE inhibitor, quinapril, on atherosclerotic plaque growth and lymphocyte invasion induced by MDV infection and hypercholesterolemia. HMGCoA reductase inhibitors [26–30]and ACE inhibitors [31–34]have been proven to reduce cardiovascular events in clinical trials. Both drugs decrease balloon injury induced atherosclerosis in animal models [35–42], but neither agent has demonstrated efficacy for prevention of restenosis in clinical studies [43–46]. HMGCoA reductase inhibitors do, however, reduce coronary vasculopathy post transplant [30]and preliminary studies with ACE inhibitors also suggest benefit in transplant vasculopathy, suggesting possible efficacy in virus associated atherosclerosis development. Both HMGCoA reductase and ACE inhibitors have also been reported to alter monocytic cell invasion, but effects on lymphocyte infiltration in the arterial wall have not been extensively studied. There have been no prior studies on the effects of current therapeutic approaches on virus-induced accelerated atherosclerosis or vascular lymphocytic infiltrates, either in the clinical setting or in animal models.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Marek's disease virus infection
In total 120 new born chicks (one day old) were inoculated with 500 cfu of the MD5 strain of Marek's disease virus (American Type Culture Collection Atlanta, Georgia); 250 cfu via intratracheal inoculation and 250 cfu via intraperitoneal injection [47]. In Study 1 the effects of pravastatin on virus-induced plaque growth were assessed in 60 chicks on normal (ND-30 chicks) and 2% cholesterol in 10% peanut oil (CD-30 chicks) diets. After 90 days each group (ND or CD) of surviving chicks was further divided into groups for treatment with either pravastatin or saline. In Study 2, chicks were maintained on CD for the duration of the study. At 90 days post infection the surviving chicks in Study 2 were divided into three groups for treatment with pravastatin, quinapril, or saline control.

In both studies, MDV infected chicks were followed after treatment was started until premature death secondary to lymphoma or to a total follow up period of 180 days post MDV infection. Chicks were monitored for signs of paralysis, decreased feeding, or generalized weakness and sacrificed early if demonstrating signs of distress. Fresh MDV isolates from the American Type culture collection were used for both studies to avoid potential altered virulence due to passage of the virus in tissue culture.

2.2 Treatment of Marek's disease virus infected chicks with pravastatin or quinapril
Study 1: of the 60 MDV infected chicks, 14 ND chicks and 12 CD chicks died secondary to MDV induced lymphoma within three months (p=0.60). At three months post infection, surviving chicks were started on pravastatin (Bristol Myers Squibb Pharmaceutical Research, Montreal, Quebec) at 0.8 mg/kg/day orally or placebo (saline) and followed for three months (8 ND chicks treated with pravastatin, 8 ND chicks treated with saline, 11 CD chicks treated with pravastatin, and 7 CD chicks treated with saline). Pravastatin or saline was given on a daily basis in a 0.25 ml saline suspension administered orally via dropper.

Study 2: 16/60 MDV infected rooster chicks died prior to the starting date for treatment. At 90 days, the 44 rooster chicks surviving the MDV infection were started on one of three treatment regimes given orally by dropper daily as follows; 0.25 ml saline (14 roosters), 0.8 mg pravastatin per kg bird weight in 0.25 ml (15 roosters), and 0.8 mg per kg bird weight quinapril (Parke Davis, Ann Arbor, Michigan) in 0.25 ml (15 roosters). After 21 days increased mortality was noted in the quinapril group and the dose of quinapril was subsequently reduced to 0.08 mg per kg body weight per bird. Chicks were weighed twice weekly and the dose adjusted according to weight.

2.3 General animal care
The chicks were maintained in a biohazard containment facility and were treated according to the guidelines of the Canadian National Committee on Animal Care and the University of Alberta Laboratory Animal Services guidelines which conform with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH Publication No. 85-23, revised 1985). Roosters displaying signs of severe infection and morbidity were sacrificed early with 3.0–4.0 ml of euthanyl (344–640 mg/kg of pentobarbitol per kg; MTC Pharmaceuticals, Canada Packers Inc., Cambridge, Ontario). Characteristic signs of MDV induced lymphoma were recorded after infection in both studies.

2.4 Histological and morphometric analysis
In Study 1, aortic specimens from three areas were excised and formalin fixed: the thoracic aorta from the left subclavian artery to the coeliac axis, the abdominal aorta from the coeliac axis to the iliac arteries, and the carotid arteries. In Study 2 the femoral arteries were examined in place of the abdominal aorta due to the fact that more atherosclerotic plaque growth was detected in the thoracic aorta and large aortic branches than in the abdominal aorta in Study 1. In Study 2, specimens were also taken from the heart, liver, spleen, and femoral nerve for histological analysis in order to assess the extent of MDV induced lymphoma.

A total of 82 arterial sections and 20 myocardial specimens for Study 1 and 192 arterial, 21 myocardial, 21 kidney, 26 liver, and 20 femoral nerve specimens for Study 2 were harvested for histological analysis. Specimens were fixed in 0.10 (v/v) sodium phosphate buffered formalin, processed, impregnated and embedded in paraffin and cut into 5 µm sections by microtome as has been previously described [48]. Sections were then stained with hematoxylin and eosin and examined by light microscopy. In Study 1, 14 sections were taken from each of the arterial specimens and 4 sections were cut from the myocardial specimens. In Study 2, 4 sections from each arterial and organ specimen were stained. The section with the largest plaque was used for morphometric analysis of the plaque area and thickness in each study. The mean area for the arterial sections taken from each bird was calculated and used for subsequent statistical analyses. All specimens, arterial and other, were independently examined by a veterinary pathologist, blinded to treatment, for evidence of MDV infection induced lymphocytic cell infiltrates and atheromatous plaque development. The MDV associated changes were graded on a scale of 0–3 by the pathologist based upon the degree of lymphoma related cellular infiltration and proliferation.

Initially intimal area and thickness were measured with a Nikon Optiphot-Labophot drawing device attached to a Nikon Labophot-2 model light microscope (Nikon, Nippon Kogaku K.K., Tokyo Japan). Plaque area was then measured with a Jandell Scientific Sigma Scan program and a digitizing Summa sketch pad coupled to a MacIntosh IIcx computer (MacMeasure software) as previously described [47, 48]. Later measurements were made with a Sony Power HAD 3CCD color video camera attached to a Zeiss Axioskop connected to the Empix Northern Eclipse trace application program (Empix Imaging Inc., Mississauga, Ontario). Each system was calibrated to the microscopic objective. Only one system was used for each series of measurements to avoid variability inherent in individual morphometric systems. In Study 2, both morphometric analysis systems were used for comparative purposes. The presence and area of lymphocytic and foam cell (vacuolated fat filled cells) infiltrates into the intima, media and adventitia were also noted and measured using this morphometric system. Fibrosis, thrombosis and calcification were not seen and were not measured in these studies. Intimal thickness was also measured by ocular micrometer.

2.5 Serum cholesterol
Serum and arterial cholesterol levels were measured in each group in Study 1 where chicks on normal diet and high cholesterol diet were assessed after treatment with either control saline or pravastatin. Cholesterol was measured using standard cholesterol assay kits (Sigma Chemical) as previously described [48, 49].

2.6 Statistics
The significance of measured differences in plaque area, plaque thickness, and cellular infiltrates on morphometric analysis were assessed by analysis of variance (ANOVA) and Student's t test analysis after drug treatment and on each dietary regimen. The mean for all plaque area and thickness measurements for individual birds in each study was calculated. The mean values for the measurements on each rooster were used for all subsequent statistical analyses. Differences in survival and potential correlations between length of treatment and/or survival time after treatment was assessed within treatment groups by Kaplan Meier survival analysis as well as Logrank and Chi square analysis.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Atherosclerotic plaque development in MDV infected chicks
Fig. 1 (Panels A and B) show typical areas of fatty cellular proliferation in a cholesterol fed MDV infected chick in Study 1 with near occlusion of the carotid arterial lumen. The mean intimal area and thickness was decreased after pravastatin treatment in the cholesterol fed rooster chicks (Fig. 1C) but not in pravastatin treated chicks on normal diets (Fig. 2A). Plaque thickness was measured as 0.046±0.011 mm and 0.013±0.005 mm respectively after control saline and pravastatin treatment for cholesterol fed chicks (p<0.03) and 0.029±0.011 mm and 0.025±0.008 mm respectively after control saline and pravastatin treatment for chicks on normal diet. A significant reduction in intimal area was also observed in Study 2 with pravastatin treatment and with quinapril treatment (Fig. 1D) after two months of treatment (Fig. 2A, B).


Figure 1
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  		Fig. 1 Hematoxylin and eosin stained sections of the carotid artery taken from MDV infected chicks after 90 days treatment, 180 days post infection. IEL, internal elastic lamina; MP, macrophage; L, lymphocytoid cells. Panel A, a large plaque producing near complete occlusion in a cholesterol fed rooster chick treated with control saline. (magnification 120x). Panel B, a higher power view of the same fatty plaque seen in Panel A demonstrating the lacy appearance of macrophage cells (foam cells) interspersed with proliferating cells in the intimal layer (magnification 460x). Panel C, a carotid artery section from a pravastatin treated, cholesterol fed rooster demonstrating a reduction in fatty plaque area in Study 1 (magnification 460x). Panel D, a carotid artery section taken from a quinapril treated, MDV infected rooster chicks demonstrating a reduction in plaque area in Study 2 (magnification 460x).

 

Figure 2
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  		Fig. 2 Panel A, bar graphs of mean plaque area±S.E. in arterial specimens taken from chicks in Study 1 on normal poultry diet (ND) and cholesterol fed (CD) after treatment with either saline control or pravastatin are shown (82 arterial specimens from 34 chicks). The mean plaque areas were measured on arterial specimens taken from chicks treated for 60 days or more with either saline or pravastatin. Panel B, scattergraph demonstrating the mean plaque area for arterial specimens taken from MDV infected birds and the corresponding lifespan and duration of treatment in Study 2 (95 arterial specimens from 44 chicks that survived past the start of the treatment at 3 months of age). An increase in plaque area becomes apparent in the saline control treated birds after 60 days treatment. Circle, control saline; diamond, pravastatin; triangle, quinapril. Panel C, Bar graphs of the mean area±S.E. of intimal (dotted bar); medial (striped bar) and adventitial (crossed bar) infiltration in the arterial wall in chicks in Study 2 after treatment (all treatment times) with either control saline, pravastatin, or quinapril. Intima, dotted bar; media, striped bar; adventitia, crossed bar. Panel D, bar graphs are shown demonstrating that there is no detectable significant difference in the area of lymphocyte infiltration shown as mean ±S.E. in any of the organs examined (26 liver, 21 kidney, 21 myocardium, or 20 peripheral nerve sections) after treatment with saline, pravastatin, or quinapril treatment. M, myocardium (solid bar); L, liver (right striped bar); K, kidney (crossed bar); N, nerve (left striped bar).

 
The intimal hyperplasia seen after MDV infection was characterized by a large number of foam cells with fat filled cytoplasm in the cholesterol fed rooster chicks (Fig. 1B). More fatty intimal hyperplasia was detected in the carotid arterial specimens than in the thoracic aorta. Minimal plaque growth was detectable in the abdominal aorta. Thrombosis, fibrosis, and calcification were not detected in this study. There is intimal hyperplasia and cellular infiltration visible in the MDV infected birds after less than 60 days of treatment but the differences in intimal hyperplasia are much less pronounced and did not reach significance for this shorter treatment time for either pravastatin or quinapril on comparison with the controls. The greatest decrease in plaque area is seen after 60 days treatment (Fig. 2B, p<0.001). No significant reduction in intimal area was detected in chicks treated for less than 60 days with either drug (p=0.87 by ANOVA). There was no statistically significant difference between the plaque area measured in chicks treated for less than one month (0.363±0.107 mm2) on comparison with arterial specimens taken from chicks that died prior to starting treatment (0.467±0.143 mm2, p=0.22) indicating that the two drugs had minimal effect on plaque growth after less than 30 days treatment.

3.2 MDV associated lymphocytic cell infiltrates in the arterial wall
In order to assess the extent of lymphocytic cellular invasion into the arterial wall, infiltrates in the intimal, medial and adventitial layers were examined. Lymphocytic infiltrates were detected by morphology (small oval mononuclear cells with round nuclei) on hematoxylin and eosin stained sections. Lymphocytes were seen in the medial and adventitial layers (Fig. 2C, Fig. 3A) more often than in the intimal layers. Care had to be taken to distinguish nucleated erythrocytes from lymphocytes, but these cells can be differentiated by the characteristic erythrocyte cellular shape with pointed ends and the distinctive red pigmentation of the erythrocyte. In the intimal layer cellular adhesion to the endothelial surface was seen more frequently than actual invasion in the intimal layer (Fig. 1C, data not shown) but did not vary with the treatment regimen.


Figure 3
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  		Fig. 3 Hematoxylin and eosin stained section of the arterial wall in a MDV infected rooster chick demonstrating lymphocytic infiltrates in the medial and adventitial layers of a carotid artery (Panel A), the myocardium (Panel B), and the kidney (Panel C). L, lymphocytoid cell infiltrate; SMC, smooth muscle cells in media (magnification 460x).

 
There was no correlation detected between plaque growth and lymphocytic invasion in the arterial wall in any of the layers in either study when assessing all specimens or after assessment of groups treated for less than 60 days or for 60–90 days with either drug (Fig. 2C). The mean area of infiltrate in the intima, media, and adventitia respectively after 60 to 90 days treatment was 0.035±0013 mm2, 0.012±0.008 mm2, 0.036±0.01 mm2 for control treatment, 0.008±0.005 mm2, 0.03±0.01 mm2, 0.005±0.003 mm2 for pravastatin, and 0.023±0.007 mm2, 0.005±0.005 mm2, 0.026±0.019 mm2 for quinapril (p=0.37 by ANOVA). There was some increase in the infiltrates seen in the quinapril treated chicks but this did not reach statistical significance. Foam cell area in the intima was also not significantly reduced by either pravastatin (0.024±0.01 mm2) or quinapril (0.003±0.003 mm2) infusion on comparison with saline control (0.014±0.007 mm2) (p=0.26 by ANOVA) (Fig. 3A).

3.3 Marek's disease virus associated lymphoma development
Typical cellular infiltrates were seen in the liver, kidney, myocardium, and femoral nerves of the chicks in Study 2. The size of the lymphocytic infiltrates was similar in all the birds both prior to and after treatment with either pravastatin or quinapril in the myocardium (Fig. 3B) (p=0.59), kidney (Fig. 3C) (p=0.18), liver (not shown) (p=0.89), or femoral nerve (not shown) (p=0.83) on comparison with saline control treatment. The bar graphs in Fig. 2D demonstrate that there is minimal change in the area of infiltrate detected with each of the treatments. There was also no significant difference in lymphocyte infiltration in any of the organs examined whether the duration of treatment was less than 60 days or for 60–90 days (confirmed by blinded analysis by a veterinary pathologist, data not shown).

3.4 Cholesterol levels
In a previously reported pilot study, the development of MDV associated plaque was examined and correlated with arterial wall cholesterol levels [48, 49]. Aortic specimens from infected birds fed a normal poultry diet had a demonstrated increase in cholesterol levels but not in triglyceride when corrected for total protein [47, 49]. This result is consistent with prior work where an increase in cellular cholesterol levels was reported with MDV infection.

In the initial pravastatin study (Study 1) serum cholesterol levels were measured in the roosters on normal poultry diet and high cholesterol diet. The serum cholesterol was higher in the cholesterol fed roosters (15.2 mg/dl) and was reduced by pravastatin in the cholesterol fed birds (9.6 mg/dl), as expected, but not in the birds on a normal poultry diet (1.3 mg/dl for both saline and pravastatin treated chicks).

3.5 Mortality
In the first study 6/7 (85.7%) of the cholesterol fed chicks treated with saline died after three months treatment. 4/11 (63.6%) of the chicks treated with pravastatin on high cholesterol diet died in the same three-month treatment period (p=0.055). 8/8 (100%) of the chicks in the saline treated and 4/8 (50%) of pravastatin treated chicks on normal poultry diets died during the three months of treatment (Fig. 4A) (p<0.02). Taken together, e.g. both birds fed a normal diet or cholesterol fed, this represents an overall 36% reduction in mortality in pravastatin treated rooster chicks whether fed cholesterol or normal diets (average mortality for control treatment was 92.9%, average mortality for pravastatin treated chicks was 56.8%) in Study 1 (p=0.22).


Figure 4
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  		Fig. 4 Analysis of the effects of pravastatin and quinapril on survival of MDV infected chicks from 90 to 180 days post infection. Panel A, bar graph demonstrating an apparent reduction in percent mortality in Study 1 with pravastatin treatment in MDV infected chicks on a normal poultry (ND) diet (p<0.02) or high cholesterol (CD) diet (p=0.055). Panel B, Kaplan–Meier cumulative survival plot for all control (circle), pravastatin (diamond), and quinapril (triangle) treated birds on a high cholesterol diet. There was no significant difference in survival after treatment with either pravastatin or quinapril.

 
In Study 2, there was no correlation (p=0.65 by Kaplan Meier survival analysis) between survival duration and either pravastatin sodium or quinapril treatment (Fig. 4B). The fact that there was no overall effect on mortality on comparison of pravastatin treatment and controls in Study 2 indicates that the initially observed decrease in mortality with pravastatin treatment in Study 1 was not reproduced in the second study.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Although viral infections have been implicated in the development of atherosclerosis, the extent to which viral infections alter atherosclerotic plaque growth and the effects of viral infection on vascular responses to anti-atherogenic agents are not known. We have detected significant reductions in plaque growth (Figs. 1–3GoGo) in a model of Marek's disease virus infected, cholesterol fed chicks after treatment with both a HMGCoA reductase inhibitor, pravastatin, and an ACE inhibitor, quinapril (Figs. 1 and 2Go). The results of this study indicate that these two drugs were able to inhibit plaque growth in cholesterol fed roosters in the presence of a prior viral infection known to induce atherosclerosis development. The use of a high cholesterol diet to speed development of atherosclerotic lesions prevents, however, analysis of the relative contributions of viral infection and hypercholesterolemia in plaque growth and its treatment.

MDV infection produces a severe and extensive lymphocytic invasion of multiple organs as well as the arterial vasculature [17–19]. An analysis of the effects of pravastatin and quinapril on lymphocytic cellular invasion did not reveal a correlation between treatment and plaque formation. However, an inhibition of monocyte or lymphocyte cellular activation acting as an early stimulus to cellular invasion and proliferation cannot be excluded. Both the HMGCoA reductase inhibitors and the ACE inhibitors have been demonstrated to alter leukocyte, specifically monocyte/macrophage, activation, albeit by differing mechanisms. The HMG CoA reductase inhibitors have been postulated to reduce monocyte and smooth muscle cellular invasion of the intima, potentially by reducing lipid oxidation products and macrophage activation [50]. Pravastatin has, however, no reported effect on smooth muscle cellular or lymphocyte migration or proliferation. The most likely mechanism for the reduction in plaque size seen with pravastatin treatment is therefor a reduction of serum cholesterol levels. ACE inhibitor therapy with quinapril has been demonstrated to improve endothelial dysfunction in patients with coronary artery disease [32]and to reduce intimal hyperplasia in several animal models of atherosclerosis [33, 35–37, 40, 51]. Other ACE inhibitors, Captopril [37], fosinopril [37], perindopril [36], and cilazapril [35], have been reported to reduce the accumulation of macrophages in the intima of hypercholesterolemic and hypertensive animal models. Cilazapril has also been found to decrease smooth muscle cellular proliferation after balloon injury of the rat carotid artery [35].

This study has also allowed us to examine the validity of Marek's disease virus infection in cholesterol fed roosters as a model for accelerated virus-induced atherosclerosis development. MDV infection is one of the few well characterized models of virus induced atherosclerosis producing an accelerated atherogenic process in the larger arterial branches and the aorta. We have detected reproducible atherosclerotic plaque development in this model with associated extensive foam cell formation and lymphocytic infiltrates. We have also detected significant decreases in plaque growth in this avian model with the administration of a HMGCoA reductase inhibitor and an ACE inhibitor indicating similar responses as seen with prior studies in mammalian models and clinical trials. There remain, nevertheless, concerns inherent in the use of this model, specifically the use of cholesterol feeding to accelerate plaque growth, the presence of an invasive lymphoma with associated high mortality, and the physiologic differences present in avian and mammalian models.

Cholesterol feeding is used to increase the rate and extent of plaque growth in MDV infected chicks, as originally reported by Fabricant et al. [17, 19, 21]. Previous reports on models of balloon injury and cholesterol feeding in White Leghorn roosters [48], as well as a study comparing virus infected rooster chicks with non-infected chicks [49]have shown that cholesterol diet alone produces a much reduced atherosclerotic plaque development on comparison with viral infection or balloon injury combined with a high cholesterol diet [48, 49]. The initiation of atherosclerotic plaque growth by both virus infection and cholesterol diet, although necessary for rapid production of plaque, does however make differentiation of the effects of cholesterol diet and viral infection difficult, as with most currently used balloon injury models. HMGCoA treatment, pravastatin in this study, most likely reduced plaque growth through a reduction in serum cholesterol [50]. Quinapril has been found to reduce serum cholesterol levels in hyperlipidemic patients [52]which may decrease macrophage activation. The doses of quinapril used in this study were low on comparison with this clinical study, but reduction in serum cholesterol and thus monocyte activation cannot be excluded as the mechanism by which quinapril decreased atheroma development.

HMGCoA reductase inhibitors, simvastatin, pravastatin and lovastatin, have been reported to decrease mortality as well as the number of cardiac events in patients with moderate to high serum levels of cholesterol [26–29]. Pravastatin reduces episodes of cardiac rejection, ischemic heart disease, incidence of coronary vasculopathy, and mortality in cardiac transplant patients [30]. ACE inhibitors reduce the incidence of myocardial infarction and mortality in patients with low ejection fractions [33, 34]. This ACE inhibitor associated reduction in mortality may be in part due to reduced atherosclerotic plaque growth that has been detected after balloon induced intimal injury [35–39]and after transplant [40]in animal models. ACE inhibitor treatment has also been demonstrated to reduce transplant vasculopathy in preliminary clinical trials [40]. We have detected a reduction in plaque growth in MDV infected chicks, but no reproducible reduction in mortality. Thus this model only partially mirrors the effects of these drugs as reported in clinical trials. The invasive lymphoma seen in MDV infection has an associated high mortality which may alter vascular responses and therapeutic efficacy and may prevent the detection of a reduction in mortality associated with the decreased atherosclerosis development produced by each drug. The effects of each drug on atherosclerotic plaque growth might also be attributed to differences in the intensity of viral infection and comorbidity, or the length of time individual chicks were treated. In order to control for variations in viral infectivity and disease severity, these studies were randomized for treatment. An initially higher dose of quinapril appeared to have an adverse effect on the MDV infected chicks and did increase mortality significantly (p<0.003). After the quinapril dose was reduced the side effects were diminished and the initial increased mortality was immediately reversed with the lower dose as seen in the survival curve (Fig. 4B).

Avian models differ physiologically from mammalian models in that the erythrocytes are nucleated and the platelets more closely resemble megakaryocytes and are again nucleated [53]. The atherosclerotic plaque that develops after balloon injury [47], MDV infection [17–19, 21], or in cholesterol fed roosters, however, resembles human atheroma in that a wide range of plaque composition is seen with fatty, fibrotic, calcific and thrombotic areas in some avian models [48, 49].

MDV infection in cholesterol fed rooster chicks may provide a valid model for the study of virus associated injury and atherogenesis, but further studies will be necessary to clarify the relative contributions of viral infection and hypercholesterolemia to plaque development in this model. This study indicates that HMGCoA reductase inhibitors and ACE inhibitors should be considered for further study as adjunctive therapy for transplant associated accelerated atherosclerosis development.

Time for primary review 35 days.


   Acknowledgements
 
Alexandra Lucas is a Scientist at the Robart's Research Institute and the Departments of Medicine and Microbiology and Immunology at the University of Western Ontario. This research was supported in part by a grant from the Alberta Heart and Stroke Foundation and a grant from Squibb Pharmaceuticals. Squibb and Park Davis supplied the drugs for testing in the MDV infected avian model. We would like to thank Marita Lundstrom Hobman and Dean Kolodziecjzyk for their help in proofing this manuscript. We would also like to thank Fran Plumb for helping with the typing of this manuscript.


   Notes
 
1 Department of Medicine, University of Alberta, Edmonton, Alberta (Institution at which this work was performed). Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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