A 6 week course of azithromycin treatment has no beneficial effect on atherosclerotic lesion development in apolipoprotein E-deficient mice chronically infected with Chlamydia pneumoniae

E. Blessing{dagger}, L. A. Campbell, M. E. Rosenfeld, B. Chesebro{ddagger} and C.-C. Kuo*

Department of Pathobiology, University of Washington, Seattle, WA 98195, USA


* Corresponding author. Tel: +1-206-543-8689; Fax: +1-206-543-3873; Email: cckuo{at}u.washington.edu

Received 1 October 2004; returned 7 February 2005; revised 9 February 2005; accepted 17 March 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To evaluate whether antimicrobial chemotherapy prevents acceleration of atherosclerotic lesion development induced by infection with Chlamydia pneumoniae.

Methods: ApoE-deficient mice which develop hyperlipidaemia and atherosclerosis spontaneously were inoculated intranasally with C. pneumoniae. Animals were treated with azithromycin for 6 weeks after the third inoculation and the atherosclerotic lesion areas in the aortic sinus were measured by computer-assisted morphometry.

Results: At 12 weeks post-infection, infected untreated animals developed significantly larger lesion areas compared with sham-inoculated controls (8.7 x 104±2.3 x 104 µm2 versus 5.6 x 104±2.4 x 104 µm2). However, there were no differences in lesion size of infected mice treated with azithromycin in comparison with untreated infected controls (11.0 x 104±3.0 x 104 µm2 versus 8.7 x 104±2.3 x 104 µm2).

Conclusions: Antibiotic treatment against C. pneumoniae has no beneficial effects on hyperlipidaemia-induced atherosclerosis accelerated by C. pneumoniae in a mouse model.

Keywords: C. pneumoniae , atherosclerosis , hyperlipidaemia , treatment , prevention


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An association of Chlamydia pneumoniae with atherosclerosis has been well established by seroepidemiology,1 detection of the organism in atherosclerotic lesions2 and animal models.36 Experiments in mouse models have shown that C. pneumoniae promotes atherosclerosis in hyperlipidaemic46 but not in normolipidaemic7 mice. The mechanisms by which C. pneumoniae promotes atherosclerosis are yet to be defined. As C. pneumoniae infection contributes to atherosclerosis, the risk of atherosclerosis could potentially be reduced by treatment with macrolide antibiotics that are known to be effective against C. pneumoniae. The potential protective effects of this type of antibiotic have previously been evaluated in animals3,6 and humans.8 However, these studies have yielded variable results. Therefore, the purpose of this study was to evaluate a long-term treatment of chronic C. pneumoniae infection with azithromycin on atherosclerotic lesion development in hyperlipidaemic apolipoprotein E (apoE)-deficient mice, which develop atherosclerosis spontaneously on a normal chow diet.4 The study design took into consideration the epidemiological observations of age of C. pneumoniae infection and development of hyperlipidaemia and atherosclerosis in humans. Seroepidemioloical studies have shown that C. pneumoniae infections are rare in children under 5 years of age.9 Age specific incidence of infection has shown that everyone gets infected between the age of 5 and 14. Thus, first infection with C. pneumoniae occurs at the age hyperlipidaemia and atherosclerosis have not fully developed. Therefore, in this study antibiotic treatment was started after chronic infection had been established in young mice in which early atherosclerotic lesions are forming.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental animals

Eight-week-old pathogen-free male apoE–/– mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed under modified specific pathogen free conditions in filter-top cages (4/cage). Routine checks did not reveal any exogenous infection during the period of the study. Mice were fed with a standard chow diet (Harlan Teklad, Madison, WI, USA) and water ad libitum throughout the study. The study protocol was approved by the University of Washington Institutional Animal Care and Use Committee.

Inoculation and treatment

Mice were sedated by intraperitoneal injection of a mixture of ketamine (Fort Dodge Laboratories, Shenandoah, IA, USA) and xylazine (Lloyd Laboratories, Shenandoah, IA, USA). To induce chronic C. pneumoniae infection in the aorta, mice were inoculated intranasally with 3 x 107 inclusion forming units of purified C. pneumoniae (strain AR-39) organisms at 8, 9 and 10 weeks of age.10 Control mice were sham-inoculated with sterile PBS. The inoculated and sham-inoculated animals were further divided into treatment and no-treatment groups. The treatment regimen consisted of administration of azithromycin (Zithromax; Pfizer Pharmaceuticals, Inc., Groton, CT, USA) at 30 mg/kg body weight by intramuscular injection into the gluteal muscle at days 3, 4 and 5 after the 3rd inoculation and once a week for 5 weeks thereafter. Sham injections were performed with sterile saline. Susceptibility of AR-39 to azithromycin has been tested in vitro and the MIC was determined to be 0.5 mg/L.11 The study design is illustrated in Figure 1.



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Figure 1. Experimental design of two experimental protocols conducted in the study.

 
Tissue collection

Necropsies were performed at 16 and 20 weeks of age (8 and 12 weeks post first inoculation). Mice were sedated with a mixture of acepromazine maleate (15 mg/kg; Fermenta Animal Health Co., Kansas City, MO, USA) and ketamine HCl (150 mg/kg; Phoenix Pharmaceutical Inc., St Joseph, MO, USA). Blood was collected by exsanguination from the femoral arteries at necropsy. The heart and aorta were perfusion fixed with 10% buffered formalin administered through the left ventricle. The lungs, heart and thoracic aorta with its main branches attached were dissected intact. The aorta was separated from the heart, and the base of the heart was frozen in OCT medium (OCTTM; Sakura Finetek, Torrance, CA, USA), and sectioned on a cryostat at the level of the aortic sinus.5 Once the atrioventricular valves were identified, 8 µm sections were taken and mounted on gelatin-coated slides. Sections of the aortic sinus were collected up to the point where the valves disappeared. Every other section throughout the sinus was used for atherosclerotic lesion analysis.

Quantification of lesion area

The cross-sectional area of an atherosclerotic lesion was determined in 15 Oil Red O stained sections per animal by computer-assisted morphometry (Optimas 5.2 software; Optimas Corp., Bothell, WA, USA) and averaged. Measurements were done in a blinded fashion with the investigators unaware of the treatment groups.

Serology and blood cholesterol measurement

Plasma was separated from heparinized blood and frozen at –70°C for serology and lipid measurements. C. pneumoniae-specific IgG and IgM antibody titres were determined by the micro-immunofluorescence test.9 Total plasma cholesterol was measured using a commercial enzymic test kit (Sigma, St Louis, MO, USA).

Statistical analysis

Data are expressed as means ± SD. Group data were analysed by the unpaired Student's t-test. A value of P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical response and lipid profiles

Clinical signs of infection included increased respiratory rate and nasal and ocular discharge. The symptoms were most severe within the first few days after the first and second inoculation. These clinical symptoms were resolved within 2 weeks after the third inoculation. No adverse reactions were observed following antibiotic treatment. There were no significant differences in body weights or cholesterol levels between any of the groups at the time of necropsy (data not shown).

Serology

All infected mice developed IgG antibodies against C. pneumoniae. The titres ranged from 1:128 to 1:512. Antibiotic treatment did not significantly reduce the antibody titres. No IgM antibodies were detected. All of the control mice were antibody negative (< 1:8).

Quantification of lesion area

There were no significant differences in lesion areas between the infected and uninfected mice at 8 weeks post-infection either in the untreated group (5.0 x 104±3.0 x 104 µm2, n=15 versus 4.1 x 104±1.6 x 104 µm2, n=10; P > 0.05) or treated group (5.5 x 104±3.4 x 104 µm2, n=15 versus 4.8 x 104±1.8 x 104 µm2, n=9; P > 0.05) (Figure 2). At 12 weeks post-infection, the infected untreated animals developed significantly larger lesion areas compared with controls (8.7 x 104±2.3 x 104 µm2, n=14 versus 5.6 x 104±2.4 x 104 µm2, n=8; P=0.026) as did the infected treated animals versus controls (11.0 x 104±3.0 x 104 µm2, n=15 versus 6.0 x 104±1.5 x 104 µm2, n=8; P=0.003) (Figure 2). However, there were no differences in lesion size of infected mice treated with azithromycin in comparison with untreated infected controls (11.0 x 104± 3.0 x 104 µm2, n=15 versus 8.7 x 104±2.3 x 104 µm2, n=14; P 0.5) (Figure 2).



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Figure 2. Effects of azithromycin treatment on atherosclerotic lesion development in apoE–/– mice. This figure shows that Chlamydia pneumoniae infection accelerated the progression of atherosclerotic lesion development and that treatment with azithromycin did not prevent the exacerbation of lesion progression by infection. Bar graphs indicate mean values ± SD; n = number of animals; *P < 0.05 versus controls, **P < 0.005 versus controls.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study confirmed our previous studies that C. pneumoniae infection in hyperlipidaemic mice accelerates the progression of atherosclerosis.4,5 However, a 6 week course of treatment with azithromycin at 30 mg/kg by intramuscular injection starting 3 days after the third weekly inoculation with C. pneumoniae did not reduce the acceleration of lesion development by C. pneumoniae infection in hyperlipidaemic apoE–/– mice. Whether a 6 week course of treatment is long enough to be effective is not known. However, considering that the life span of mice is 2.5 years, 6 weeks of treatment represents 4.6% of the life span of mice, which would be equivalent to 3.3 years of treatment in humans.

Our results are similar to those reported by Rothstein et al.,6 using the same mouse model but with a shorter treatment regimen. In their study, mice were inoculated with C. pneumoniae twice at 2 week intervals and azithromycin was administered orally at 24 mg/kg once a week for 2 weeks starting 2 weeks after the second inoculation. No reduction in lesion development induced by C. pneumoniae was observed at either 10 or 14 weeks post-infection.

In contrast, in New Zealand white rabbits, weekly treatment by intramuscular injection of 30 mg/kg of azithromycin for 7 weeks prevented the acceleration of intimal thickening induced by C. pneumoniae in rabbits given a diet supplemented with a small amount (0.25%) of cholesterol.3 In this model, animals were inoculated three times at 3 week intervals and the antibiotic treatment was started immediately after the final inoculation.

Human therapeutic trials with macrolides have also yielded variable results.8 Of eight small clinical trials, three trials showed positive results, four negative results and one equivocal results.8 A large-scale randomized control trial of 7747 adults who had previous myocardial infarction with a 3 month course of azithromycin [Weekly Intervention with Zithromax for Atherosclerosis and Related Disorders (WIZARD)] was recently completed and reported.12 This study showed that this treatment regimen did not reduce the overall clinical sequelae of coronary heart disease after a median of 14 months follow-up. However, a beneficial effect was observed with a 30% reduction in the incidence of death or non-fatal re-infarction at 6 months after stopping treatment. The ACES (Azithromycin and Coronary Events Study) study has just been completed in 2004.8 In this study, patients were treated with 600 mg of azithromycin orally once a week for 1 year. No significant clinical benefit in the secondary prevention of coronary heart disease events has been observed. (Presented by J. T. Grayston at the Fifth European Society for Chlamydia Research, September 1–4, 2004, in Budapest).


    Footnotes
 
{dagger} Present address. Innere Medizin III, Ruprecht-Karl-Universitat Heidelberg, Heidelberg, Germany. Back

{ddagger} Present address. Department of Anesthesia, University of California at San Francisco, San Francisco, CA, USA. Back


    Acknowledgements
 
We would like to thank Jerry Ricks, Anne Techlenburg and Amy Lee for technical assistance. This work was supported in part by a National Institute of Health grant HL-56036 and a research grant from Pfizer Pharmaceuticals, Inc.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Saikku P, Leinonen M, Mattila K et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 1988; ii: 983–6.

2 . Kuo C-C, Shor A, Campbell LA et al. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis 1993; 167: 841–9.[ISI][Medline]

3 . Muhlestein JB, Anderson JL, Hammond EH et al. Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model. Circulation 1998; 97: 633–6.[Abstract/Free Full Text]

4 . Moazed T, Campbell LA, Rosenfeld ME et al. Chlamydia pneumoniae infection accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. J Infect Dis 1999; 180: 238–41.[CrossRef][ISI][Medline]

5 . Blessing E, Campbell LA, Rosenfeld ME et al. Chlamydia pneumoniae infection accelerates hyperlipidaemia induced atherosclerotic lesion development in C57BL/6J mice. Atherosclerosis 2001; 158: 13–17.[CrossRef][ISI][Medline]

6 . Rothstein NM, Quinn TC, Madico G et al. Effect of azithromycin on murine arteriosclerosis exacerbated by Chlamydia pneumoniae. J Infect Dis 2001; 183: 232–8.[CrossRef][ISI][Medline]

7 . Blessing E, Lin T-M, Campbell LA et al. Chlamydia pneumoniae induces inflammatory changes in the heart and aorta of normocholesterolemic C57BL/6J mice. Infect Immun 2000; 68: 4765–8.[Abstract/Free Full Text]

8 . Grayston JT. Antibiotic treatment of atherosclerotic cardiovascular disease (editorial). Circulation 2003; 107: 1228–30.[Free Full Text]

9 . Kuo C-C, Jackson LA, Campbell LA et al. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 1995; 8: 451–61.[Abstract]

10 . Campbell LA, Moazed TC, Kuo C-C et al. Preclinical models for Chlamydia pneumoniae and cardiovascular disease: hypercholesterolemic mice. Clin Microbiol Infect 1998; 4 Suppl 4: S23–S32.[Medline]

11 . Kuo C-C, Jackson LA, Lee A et al. In vitro activities of azithromycin, clarithromycin, and other antibiotics against Chlamydia pneumoniae. Antimicrob Agents Chemother 1996; 40: 2669–70.[Abstract]

12 . O'Connor CM, Dune MW, Pfeffer MA et al. Azithromycin for the secondary prevention of coronary heart disease events: the WIZARD study: a randomized control trial. JAMA 2003; 290: 1459–66.[Abstract/Free Full Text]





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