Chlamydia pneumoniae Does Not Increase Atherosclerosis in the Aortic Root of Apolipoprotein E Deficient Mice

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1 Chlamydia pneumoniae Does Not Increase Atherosclerosis in the Aortic Root of Apolipoprotein E Deficient Mice Katriina Aalto-Setälä, Kirsi Laitinen, Leena Erkkilä, Maija Leinonen, Matti Jauhiainen, Christian Ehnholm, Minna Tamminen, Mirja Puolakkainen, Ilkka Penttilä, Pekka Saikku Abstract In epidemiological studies, an association between cardiovascular disease and Chlamydia pneumoniae (C pneumoniae) infection has been observed. Although C pneumoniae has been shown to be present in atherosclerotic lesions, a causal relationship between C pneumoniae infection and atherosclerosis has not been demonstrated. To study this question, we used 2 strains of apolipoprotein (apo) E deficient mice. Eight-week-old mice on an FVB background that were maintained on either a low- or a high-fat diet were infected 3 times at 1-week intervals with C pneumoniae, and atherosclerotic lesions were measured in the aortic root at 10 weeks after the primary infection. In each of the diet groups, no difference in the extent of atherosclerosis could be observed between the C pneumoniae infected and control animals. In further studies, 2 strains of apoe-deficient mice (FVB or C57BL/6J background) were infected 4 times at 3- to 4-week intervals, and the extent of atherosclerosis was analyzed 18 weeks later. The mice were kept on either a low- or a high-fat diet. The high-fat diet increased atherosclerosis, and a difference in atherosclerosis susceptibility between the mouse strains was observed. However, C pneumoniae infection did not influence lesion size in either mouse strain. On the other hand, C pneumoniae could not be demonstrated by polymerase chain reaction in any of the atherosclerotic lesions of the infected animals studied. A small decrease in serum cholesterol and triglyceride levels 3 days after the primary infection occurred, but after that no differences in serum lipid levels compared with those in noninfected animals were evident. In the myocardium of C pneumoniae infected mice, no inflammatory signs could be observed. We conclude that under the experimental conditions used, C pneumoniae infection does not accelerate atherogenic changes in the aortic root of apoe-deficient mice. (Arterioscler Thromb Vasc Biol. 2001;21: ) Key Words: C pneumoniae infection atherosclerosis apoe-deficient mice animal model Coronary heart diseases (CHDs) are the leading cause of mortality in Western societies. Atherosclerosis is associated with several risk factors, including smoking, hypertension, dyslipidemia, diabetes, positive family history, male sex, and age. However, only 50% of the CHD risk can be explained by these factors, and therefore, other risk factors must contribute. 1 One risk factor that has been proposed is chronic infection. 2 At present, several lines of evidence suggest that atherosclerosis may be regarded as a chronic inflammatory disease and that infections may play an important role in perpetuating the inflammatory status. One pathogen that has been implicated to strongly influence atherogenesis is Chlamydia pneumoniae (C pneumoniae) (reviewed by Muhlestein 3 ). This obligate, intracellular bacterium is responsible for a significant proportion of upper and lower respiratory infections, including 10% of all pneumonia in adults and 5% of bronchitis and sinuitis. 4 The prevalence of C pneumoniae antibodies in the population ranges from 50% at the age of 20 years to almost 80% in old age. 5 Besides causing acute inflammatory diseases, C pneumoniae has been found to be associated with a number of chronic diseases, including asthma, chronic obstructive pulmonary disease, and reactive arthritis. 4 Several epidemiological studies have demonstrated the association of CHD and elevated plasma C pneumoniae antibody levels (see reviews 3,6,7 ). However, this association has not been confirmed in all studies In prospective studies, only sparse or no evidence for the association has been obtained The observation that C pneumoniae can be detected in atheromatous plaques further strengthens the assumption that C pneumoniae is a causative agent of atherogenesis. 17 C pneumoniae has been shown to be present in almost 60% of atherosclerotic lesions compared with 3% in the normal vessel wall. 18 The drawback with both epidemiological studies and with those detecting C pneumoniae in the lesions is that neither can determine whether C pneumoniae really has a role in Received October 31, 2000; revision accepted November 27, From the Department of Internal Medicine (K.A.-S.), Tampere University Hospital, and the Department of Medical Biochemistry (K.A.-S.), University of Tampere, Tampere; the Department of Public Health Sciences (K.L.), University of Helsinki, Helsinki; the National Public Health Institute (L.E., M.L.), Oulu; the National Public Health Institute (M.J., C.E.), Department of Biochemistry, Helsinki; the Department of Internal Medicine and Biocenter Oulu (M.T.), University of Oulu, Oulu; the Haartman Institute (M.P.), Department of Virology, University of Helsinki, Helsinki; the Department of Clinical Chemistry (I.P.), Kuopio University Hospital, Kuopio; and the Department of Microbiology (P.S.), University of Oulu, Oulu, Finland. Correspondence to K. Aalto-Setälä, Tampere University Hospital, Department of Internal Medicine, PO Box 607, FIN Tampere, Finland. lokaaa@uta.fi 2001 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at 578

2 Aalto-Setälä et al C pneumoniae Does Not Increase Atherosclerosis in ApoE -/- Mice 579 atherogenesis or whether it is just an innocent bystander in the lesions. Studies with antibiotics have been performed but so far, only in small populations. 19,20 However, from those studies it is difficult to conclude whether the results obtained are due to an antimicrobial effect or to nonspecific, antiinflammatory effects. Large antibiotic trials are underway, 19,21 but still the crucial question as to whether bacterial infection really has a causal role in atherogenesis or in lesion stability cannot be answered by those kinds of studies. 22 To test whether there exists a causal relationship between C pneumoniae infection and atherosclerosis, we performed studies in which apolipoprotein E (apoe) deficient mice were used as a model for atherosclerosis. The causal relationship is not necessarily the same in humans as it is in animals, but at least so far, the effects of different factors on atherosclerosis in mice have been similar to what would be predicted in humans. 23 This hyperlipidemic animal model can spontaneously develop atherosclerotic lesions that, based on their morphology, are very similar to those in humans. These mice were repeatedly infected with C pneumoniae, and the initiation and progression of atherosclerosis were analyzed in the aortic root. Both sexes of mice of 2 different genetic backgrounds were used in combination with low- and highfat diets. Methods Mice ApoE-deficient mice on FVB and C57BL/6J backgrounds were used. FVB mice were kindly provided by Prof J.L. Breslow of Rockefeller University, New York, NY, and C57BL/6J mice were obtained from the Jackson Laboratories (Bar Harbor, Me). Mice were housed individually in filter-top cages and fed with either regular low-fat chow (Purina) or a high-fat diet (21% wt/wt fat, 0.15% wt/wt cholesterol, 19.5% wt/wt casein, and no sodium cholate; Harlan Teklad TD 88137) ad libitum. Both male and female mice were used. The animal experiments used in this article were approved by the ethics committee of the University of Tampere. Inoculation At 8 weeks of age, the mice were mildly sedated with methoxyflurane and inoculated intranasally with the C pneumoniae Kajaani 7 strain in a volume ranging from 20 to 40 L in sucrose-phosphate glutamic acid (SPG) chlamydial transport medium. To determine the influence of C pneumoniae on serum lipid levels, 1 set of mice was infected once with inclusion-forming units (IFU) of C pneumoniae and killed at certain time points for lipid analysis. In the first experiment to study the effect of C pneumoniae on atherosclerosis, the mice were inoculated with IFU of C pneumoniae 3 times at 1-week intervals and killed 10 weeks later. In the second experiment, the mice were infected 4 times at 3- to 4-week intervals and killed 18 weeks later. In this experiment, mice were first infected with IFU of C pneumoniae, but because 30% of the infected mice died a few days after infection and the rest of them looked very sick for a few days, later infections were performed with IFU of C pneumoniae. As controls, mice were inoculated with the same amount of SPG by following the protocol used with those receiving C pneumoniae. C pneumoniae antibody titers were analyzed by a microimmunofluorescence method with the use of elementary bodies of the Kajaani 6 strain as antigen (slides from Labsystems) and fluorescein-conjugated anti-mouse IgG as a conjugate (Sigma). Blood Collection When the mice were killed, 500 to 1000 L of blood was collected. The blood collections were performed in the morning with the animals having free access to food. Serum samples were fractionated by size-exclusion chromatography on Superose 6 HR gel-filtration Figure 1. Serum total cholesterol (A) and triglycerides (B) in C pneumoniae infected (f) and control ( ) apoe-deficient mice. Eight-week-old mice were inoculated once, and 4 to 11 mice in both groups were killed at each time point. The average values are presented (). columns, with 2 columns (Pharmacia) connected in tandem. Gelfiltration buffer was PBS, ph 7.4. Pooled serum samples (250 L) were applied to the equilibrated column at a flow rate of 0.5 ml/min, and 0.5-mL fractions were collected for further lipid analysis. Serum total cholesterol and triglycerides as well as their concentrations in the gel-filtration fractions of plasma were measured enzymatically with the use of commercial kits (cholesterol, Boehringer Mannheim catalog No ; triglycerides, GPO-Trinder, Sigma catalog No. 337-B). Quantitative Atherosclerosis Measurements Mice were killed (with CO 2 ) and then perfused with saline. Hearts were fixed in 4% phosphate-buffered formaldehyde. For the quantitative lesion assay, hearts were embedded in 25% gelatin and cryostat-sectioned at 10- to 12- m thickness, and processing and staining of the aortic root were carried out according to methods previously described by Paigen et al. 24 Quantification of lesion areas in the aortic root was performed as described earlier The sections were evaluated for oil red O staining areas by capturing the images from a camera attached to a light microscope and displayed on a computer monitor. Image analysis was performed with Image- Pro Plus software, version 3.0, in the first experiment and version 4.0 in the second experiment (Medio Cybernetics). PCR Analysis DNA was extracted from murine aortic tissues by proteinase K lysis and phenol-chloroform extraction, followed by ethanol precipitation. The extracted DNA was amplified by C pneumoniae specific nested primers in a touchdown polymerase chain reaction (PCR) as described by Tong and Sillis. 28 Amplified products were detected by agarose gel electrophoresis and ethidium bromide staining, as well as by Southern blotting and hybridization with a digoxigenin-labeled probe. Histology Assay Separate heart sections were stained with hematoxylin and eosin for the presence of inflammation in the heart muscle. 29

3 580 Arterioscler Thromb Vasc Biol. April 2001 TABLE 1. Lipid Levels in ApoE-Deficient Mice on an FVB Background Cholesterol, Triglycerides, n Low-fat diet C pneumoniae Control High-fat diet C pneumoniae * Control * Serum total cholesterol and triglycerides in apoe-deficient male mice on an FVB background maintained on low-fat chow and high-fat diets for 10 weeks were measured after 3 repeated inoculations with C pneumoniae or control SPG buffer. Data are mean SD, where n number of animals. *P when compared with low-fat diet. Statistical Analysis Plasma lipids are reported as mean SD. Comparisons between groups were made with Student s 2-tailed t test. Results To study the effect of C pneumoniae infection on serum lipid levels, we used apoe-deficient mice on an FVB background, and the mice were maintained on the (low-fat) chow diet. One group was infected once with IFU of C pneumoniae while the other served as a control. At each time point, 4 to 11 infected and control mice were used (Figure 1). Before infection, serum cholesterol values were similar in both groups ( in the infected group vs in controls; P NS). C pneumoniae infection caused a small drop in total cholesterol 3 days after TABLE 2. Lipid Levels in ApoE-Deficient Mice on an FVB Background Cholesterol, Triglycerides, n Diet Males FVB background C pneumoniae 3 High fat * Control 5 High fat C57BL/6J background C pneumoniae 4 Chow Control 5 Chow C pneumoniae 1 High fat Control 5 High fat Females C57BL/6J background C pneumoniae 5 Chow Control 5 Chow C pneumoniae 3 High fat Control 4 High fat Serum total cholesterol and triglycerides in apoe-deficient mice on FVB and C57BL/6J backgrounds were measured 18 weeks after initial inoculation with either C pneumoniae or control SPG buffer. Data are mean SD, where n number of animals. *P 0.01, P 0.001, and P when compared with values in male mice with a C57BL/6J background on a high-fat diet. P when compared with values on a low-fat chow diet. the infection (P 0.09; Figure 1A), but thereafter the serum cholesterol levels were similar in infected and noninfected animals. Also at day 3 after infection, a decrease in serum triglycerides was evident (P 0.01). Total triglyceride values differed significantly only 6 weeks (42 days) after C pneumoniae inoculation ( in infected and in control animals; P 0.02). The reduction in total cholesterol and triglyceride values 3 days after infection was due to a drop in the VLDL fraction (data not shown). Similar results were obtained in experiments in which repeated inoculations with C pneumoniae were used (Table 1). In the groups fed a high-fat diet, serum cholesterol values increased almost 5-fold while serum triglyceride levels were unaffected (Table 2). Also in mice on the high-fat diet, C pneumoniae infection did not cause any difference in plasma lipid levels compared with those in noninfected animals. It can thus be concluded that under the experimental conditions used, C pneumoniae does not significantly affect plasma lipid levels. To study the effect of C pneumoniae on the progression of atherosclerotic lesions, 2 separate sets of experiments were performed. ApoE-deficient FVB male mice were infected with C pneumoniae 3 times at 1-week intervals between inoculations. Ten control and ten C pneumoniae infected mice were kept on the chow diet, and 6 control and 4 infected mice were fed the high-fat diet. All C pneumoniae infected mice had antibodies against C pneumoniae, with titers ranging from 32 to 128 (mean, 96), whereas in the sera from noninfected mice, no antibodies were present. Serum total triglyceride and cholesterol values were not significantly different between C pneumoniae infected and control mice on either diet (Table 1). Ten weeks after the first inoculation, the mice were killed and the atherosclerotic lesions characterized. In noninfected control mice fed a chow diet, the mean aortic lesion area was m 2 while in chow-fed, C pneumoniae infected mice, the area was m 2 (Figures 2 and 3). In mice fed a high-fat diet, the atherosclerotic lesions were much more pronounced: in the control group and m 2 in the infected group (Figure 3). However, there was no significant difference in the magnitude of lesion size in C pneumoniae infected and noninfected control mice on either diet. In the next set of experiments, 2 strains of apoe-deficient mice, on FVB and C57BL/J6 backgrounds, were used (Figures 2 and 4). The animals were inoculated 4 times with 3 to 4 weeks between inoculations and killed 18 weeks after the first inoculation. All C pneumoniae infected mice had antibodies against C pneumoniae (titers ranging from 32 to 128; mean, 101), but none of the controls did. Five FVB male mice were C pneumoniae infected and 5 served as controls, and all 10 were kept on the high-fat diet. Two infected mice died after the primary infection. The mean lesion areas were and m 2 in infected and control mice, respectively (P NS). Five C57BL/6J male mice (1 died after the primary infection) were infected with C pneumoniae and 5 were kept as controls, and all 10 mice were kept on the chow diet. Five C pneumoniae infected C57BL/6J mice (4 mice died after the primary infection) and 5 controls were kept on the high-fat diet. The lesion areas in C pneumoniae infected and control mice maintained on the chow diet were and m 2, respectively, and on the high-fat diet, and

4 Aalto-Setälä et al C pneumoniae Does Not Increase Atherosclerosis in ApoE -/- Mice 581 Figure 2. Oil Red O stained sections of aortic root from C pneumoniae infected and control mice. Lesions in apoedeficient mice on an FVB background fed the chow diet (C pneumoniae infected [A)], control media [B]) 10 weeks after the primary inoculation. Lesions in apoe-deficient mice on the C57BL/6J background that were fed the chow (C pneumoniae infected [C], control [D]) and the high-fat (C pneumoniae infected [E], control [F]) diet 18 weeks after the primary inoculation m 2, respectively (Figures 2 and 4). Atherosclerosis was analyzed in C57BL/6J female mice also. Five C pneumoniae infected and 5 control female mice were kept on the chow diet and 3 infected and 4 controls, on the high-fat diet. The mean lesion areas for the chow diet fed group were m 2 for infected and m 2 for control animals. The corresponding figures for female mice maintained on the high-fat diet were and m 2, respectively (Figure 4). The atherosclerotic lesion areas in C pneumoniae infected and control mice were not significantly different. Serum lipid values did not differ significantly from each other in C pneumoniae infected and control groups (Table 2). The cholesterol-raising effect of the high-fat diet was clearly displayed in both male and female mice. However, the diet did not affect serum triglyceride levels. Even though atherosclerosis was more extensive in C57BL/6J mice than in FVB mice, on the high-fat diet FVB mice had significantly higher serum total triglyceride and cholesterol levels than did C57BL/6J mice (P ). The presence of C pneumoniae DNA was analyzed by PCR in the aortic arch in 20 C pneumoniae infected mice and 10 control mice. None of the aortic samples were positive for chlamydial DNA (data not shown). The myocardium of C pneumoniae infected and control mice was also analyzed. The overall morphology was not different in the 2 groups, and no inflammatory reactions, as determined by the absence of clusters of mononuclear inflammatory leukocytes, were observed in the myocardium or in the perivascular area of either control or C pneumoniae infected mice (data not shown). Discussion On the basis of epidemiological studies and the localization of C pneumoniae in atherosclerotic lesions, C pneumoniae has been suggested to play a role in the development of atherosclerosis. Whether it influences the initiation or progression of atherogenesis or has a role in the regulation of plaque stability was not answered by those studies. We studied the influence of C pneumoniae in a relevant animal model to follow the development of atherosclerotic lesions. ApoE-deficient mice were infected intranasally with C pneumoniae, and atherosclerotic changes were quantified in the aortic root. The animals were infected repeatedly to cause a

5 582 Arterioscler Thromb Vasc Biol. April 2001 Figure 3. Atherosclerotic lesions in C pneumoniae infected (black bars) and control (gray bars) apoe-deficient mice on chow and high-fat diets. The 8-week-old mice were inoculated 3 times 1 week apart and killed 10 weeks later. Lesion areas were quantified from the aortic root as described in Methods. Ten C pneumoniae infected (1) and 10 control (2) mice were kept on the chow diet, and 4 infected (3) and 6 control (4) mice were fed the high-fat diet. The average lesion areas are presented ( m 2 ). chronic C pneumoniae infection. At first, reinfections were performed at 1-week intervals, but with the evidence that in mice C pneumoniae can be cultivated from lungs up to 4 weeks after infection, 30 later reinfections were performed 3 to 4 weeks apart. With both infection protocols, we could not demonstrate differences in lesion areas between C pneumoniae infected and control mice. Hu and coworkers 31 demonstrated in LDL receptor deficient mice that C pneumoniae infection could have an effect on atherosclerosis but only when combined with elevated serum cholesterol. We kept the mice on both the low-fat chow diet and on a high-fat diet and could clearly demonstrate an increase in the degree of atherosclerosis when serum lipids were elevated. However, C pneumoniae infection did not affect the progression of atherosclerosis on either diet. Different mouse strains have been demonstrated to vary in their susceptibility to acquire atherosclerotic lesions, 32 and the same has been observed in apoe-deficient mice on different background strains. 33 We wanted to analyze whether 1 background would be more sensitive than another to the possible atherogenic effect of C pneumoniae. An atherosclerosis-prone background, Figure 4. Atherosclerotic lesions in C pneumoniae infected (black bars) and control (gray bars) apoe-deficient mice on FVB and C57BL/J6 backgrounds. The 8-week-old mice were inoculated 4 times 3 to 4 weeks apart and killed 18 weeks later. FVB mice (1 and 2) were males and were maintained on the high-fat diet. C57BL/J6 mice were males (3 to 6) and females (7 to 10) and kept on either the low-fat chow diet (3 and 4; 7 and 8) or the high-fat diet (5 and 6; 9 and 10). The lesion areas were quantified from the aortic root as described in Methods. The average lesion areas are presented ( m 2 ). C57BL/6J, and an atherosclerosis-resistant background, FVB, were chosen. C pneumoniae infection did not, however, accelerate atherosclerosis in either of the 2 strains. Both male and female mice were used, and no effects of C pneumoniae on atherogenesis could be observed in either sex. These findings suggest that C pneumoniae infection is not critical in the initiation or progression of atherosclerosis in this animal model. Animal models have also been used to analyze the effect of C pneumoniae on atherosclerosis. In rabbits, endothelial intimal thickening, grade III atherosclerotic lesions, and even calcification have been detected in C pneumoniae infected animals Furthermore, it has been possible to prevent C pneumoniae induced intimal thickening by administering antibiotics that are effective against Chlamydia. 35 In LDL receptor deficient mice, C pneumoniae infection was observed to increase atherosclerosis, but only 9 months after the primary infection and only on a high-fat diet. 31 The infection did not increase atherosclerosis at 6 months after infection and on the high-fat diet, and no effect was observed 6 or 9 months after infection in conjunction with a low-fat diet. In the present article, the longest time of C pneumoniae exposure was 4 months, and thus, this relatively short period could be 1 plausible explanation for the different findings in these 2 studies. However, mice 9 months of age are already quite old, and the finding with LDL receptor deficient mice would mean that even though C pneumoniae was present in those mice from the beginning, it affected the process only later in life. Moazed and coworkers 38 used apoe-deficient mice as the animal model for C pneumoniae infection and atherosclerosis. They found that C pneumoniae accelerated the atherosclerotic process significantly. In their experiments, the increase was observed even on a low-fat diet and was already detectable 8 weeks after the initial infection, when the mice were 16 weeks of age. The main difference in their experimental setup compared with ours is the aortic segment where atherosclerosis was quantified: they used the aortic arch and we used the aortic root. It is possible that the atherosclerotic process is different in these 2 locations. However, it has been shown earlier that both of these locations are sites of predilection for lesion development. 39 It is, however, possible that lesion progression is different in the 2 places and that C pneumoniae preferentially influences the process in the aortic arch. The evidence for C pneumoniae as a potential causative agent for atherosclerosis is based on serological studies and the presence of these bacteria in atherosclerotic lesions. In the current work, we found antibodies to C pneumonia in all of the infected mice, whereas none of the noninfected control mice had antibodies to C pneumoniae. However, we could not detect C pneumoniae DNA in any of the aortic samples analyzed. C pneumoniae bacterial viability was based on the fact that the animals looked sick for a few days after each infection, and when too many bacteria were accidentally used as an inoculant, as described in Methods, some of the mice died of the infection. Aortic arches as well as aortic roots have been found to be areas with the most pronounced atherosclerotic lesions. 39 Our PCR analysis was done with aortic arch samples, and thus, the negative finding cannot be explained by a lack of lesions in the tissues analyzed. The C pneumoniae strain used in our experiments was different

6 Aalto-Setälä et al C pneumoniae Does Not Increase Atherosclerosis in ApoE -/- Mice 583 from strains used by other groups. In our study, the C pneumoniae strain Kajaani 7 (a Mycoplasma-free Finnish epidemic strain) was used. 40 In the work by Hu and coworkers, the C pneumoniae strain AR-39 was used, and the presence of chlamydial antigens in atherosclerotic lesions was reported. 31 Campbell and coworkers 41 also infected mice with C pneumoniae strain AR-39, and they could detect chlamydial DNA in aortic tissues. Also, the age of the mice infected was observed to be critical in the study by Campbell and coworkers. 41 When the mice were infected at 16 weeks of age, all of the aortas examined contained chlamydial DNA, but when the infection was performed at 8 weeks of age, only part of the aortas examined contained chlamydial DNA. 41 Our mice were all 8 weeks old when they were first infected. However, in the study by Moazed and coworkers, 38 in which C pneumoniae infection increased atherosclerosis, the mice were also infected at the age of 8 weeks. The strain used in their study was AR-39. LDL receptor deficient mice used by Hu and coworkers 31 were 4 to 5 weeks of age when first infected. Based on these findings, it can be concluded that the presence of antibodies to C pneumoniae does not necessarily mean that the C pneumoniae organism would be present in atherosclerotic lesions, and there could be chlamydial strain specific differences in their ability to disseminate from lungs to other tissues such as the arterial wall. In human studies, the initiation and progression of atherogenesis are difficult to examine, and the presence of atherosclerosis usually becomes evident only after acute vascular events have occurred, ie, when advanced lesions are already present. Most epidemiological studies published thus far have found elevated C pneumoniae antibody titers to be associated with advanced atherosclerotic lesions and acute vascular events with plaque rupture, resulting in acute myocardial infarction or stroke. 3,6,7 These studies do not tell much about the role of C pneumoniae infection in atherogenesis but mainly about the final stage, with rupture of the plaque and thrombus formation. Thus, these studies suggest that C pneumoniae infection could have a crucial role in plaque stability. The presence of C pneumoniae in lesions does not prove that it has a role in atherogenesis. C pneumoniae is found in monocyte-macrophages and can be disseminated from the lungs to atheromatous plaques; thus, it could be in the lesions as an innocent bystander. 42 It is possible that when present in the lesion, C pneumoniae causes a chronic inflammation and thus perpetuates atherogenesis, but the presence of the bacterium in the lesion does not prove this hypothesis. Another possible mechanism for the association between C pneumoniae and acute vascular events is that C pneumoniae makes the plaque unstable. C pneumoniae contains lipopolysaccharide and heat shock protein 60, 43 both of which are strong inducers of matrix metalloproteinases, and thus, the presence of C pneumoniae could enhance the action of proteolytic enzymes and predispose towards plaque rupture. Our study was not designed to answer whether C pneumoniae causes atherosclerotic plaques to be unstable but rather to determine whether the infection plays a role in the initiation and progression of atherosclerosis. Mice do not normally have infarctions, even though they develop advanced atherosclerotic lesions, and currently, we do not have a good animal model for infarction. With current animal models, additional manipulations are needed if one wants to test the role of C pneumoniae infection in plaque instability and rupture. Chlamydia infections have also been associated with other types of heart diseases. 44 Bachmaier and coworkers 29 found that an outer membrane protein of C pneumoniae has strong sequence homology with a peptide from murine heart muscle specific -myosin heavy chain. Immunization of mice with the peptide from the -myosin heavy chain induces severe autoimmune heart disease. Immunizing mice with the homologous peptide from C pneumoniae induced a similar reaction, with perivascular inflammation, fibrotic changes, and blood vessel occlusion in the heart. These observations would have occurred through antigenic mimicry. We therefore analyzed the myocardium of control and C pneumoniae infected mice but could not detect any signs of perivascular inflammation or fibrosis in either group. In Bachmaier s and our work, different mouse strains were used, and this may help to explain the difference. Also, whereas Bachmaier and coworkers 29 used purified peptides that were infused into mice, in our experiments, whole live C pneumoniae bacteria were delivered intranasally into mice. In the current study, we could not demonstrate that intranasally administered C pneumoniae infection caused inflammatory reactions in the heart muscle of apoe-deficient mice. In conclusion, our experiments do not demonstrate any effect of C pneumoniae infection on the initiation or progression of atherosclerosis in apoe-deficient mice. Although seroconversion was obvious in all mice after infection with C pneumoniae, no C pneumoniae DNA could be demonstrated in aortic tissues. It is possible that the C pneumoniae strain used in our experiment does not disseminate from the respiratory system. It is also possible that the age of primary infection could be critical for the bacteria to disseminate from the lungs. Our experiments demonstrate that the presence of antibodies to C pneumoniae does not mean that the bacteria can also been found in the arterial wall. Further human and animal studies are needed to determine the molecular mechanisms underlying the reported association between C pneumoniae infection and atherosclerotic disease. Acknowledgments This work was supported by the Finnish Heart Foundation and Finnish Cultural Foundation. The technical assistance of Merja Lehtinen, Terttu Korpela, Carola Andersson-Parkkonen, and Mika Paldanius is acknowledged. References 1. Crouse JR. Progress in coronary artery disease risk factor research: what remains to be done? Clin Chem. 1984;30: Noll G. Pathogenesis of atherosclerosis: a possible relation to infection. Atherosclerosis. 1998;140(suppl 1):S3 S9. 3. Muhlestein JB. Chronic infection and coronary artery disease. Med Clin North Am. 2000;84: Kou C-C, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev. 1995;4: Wang S-P, Grayston JT. Population prevalence antibody to Chlamydia pneumoniae, strain TWAR. In: Bowie WR, Coldwell HD, Jones RP, et al, eds. Chlamydial Infections. Cambridge, England: Cambridge University Press; 1990: Saikku P. Epidemiology of Chlamydia pneumoniae in atherosclerosis. Am Heart J. 1999;138(suppl):S500 S Orfile JJ. Seroepidemiological evidence for an association between Chlamydia pneumoniae and atherosclerosis. Atherosclerosis. 1998; 140(suppl 1):S11 S15.

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