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Delivery of Chlamydia pneumoniae to the vessel wall aggravates atherosclerosis in LDLr-/- mice

A.D. Hauer, P. de Vos, N. Peterse, H. ten Cate, Th.J.C. van Berkel, F.R.M. Stassen, J. Kuiper
DOI: http://dx.doi.org/10.1016/j.cardiores.2005.07.011 280-288 First published online: 1 January 2006


Objective: The role of Chlamydia pneumoniae in atherosclerosis is still debated. In this study a novel mouse model was applied to determine the direct impact of C. pneumoniae on the arterial wall and the development of atherosclerosis.

Methods: Direct effects of C. pneumoniae on collar-induced atherosclerosis were studied after local delivery of C. pneumoniae to carotid arteries of LDL receptor-deficient (LDLr-/-) mice.

Results: The presence of C. pneumoniae in the vessel wall was quantified by RT-PCR (6.2 × 104 copies/artery) and resulted in a 2.0-fold increase in intima/media ratios (p<0.05) and a 1.7-fold increase in stenosis (p<0.05). Immunostaining revealed a 2.98-fold (p<0.01) increased macrophage content and a tendency towards lower numbers of smooth muscle cells and collagen in lesions of infected carotid arteries. Direct delivery of another respiratory pathogen, Mycoplasma pneumoniae, to the carotids did not affect size or composition of the atherosclerotic lesions. Presence of C. pneumoniae in the carotid arteries resulted within 7 days in a marked upregulation of the expression of MCP-1 (p<0.01) and ICAM-1 as determined on mRNA and protein levels. These in vivo data were in line with data obtained with in vitro infections of macrophages and endothelial cells with C. pneumoniae.

Conclusions: We conclude that C. pneumoniae in carotid arteries leads to more pronounced atherosclerotic lesions with a more vulnerable morphology and that this model is suitable to monitor direct effects of C. pneumoniae on atherogenesis.

  • Chlamydia pneumoniae
  • Atherosclerosis
  • Inflammation
  • Mouse model

Atherosclerosis is considered to be an inflammatory disease [1] and possible involvement of infectious agents has been studied. Until now most evidence indicates that Chlamydia pneumoniae may promote atherogenesis. This gram-negative bacterium, a common human pathogen, causes mild upper respiratory tract infections. The majority of people are repeatedly infected with C. pneumoniae during a lifetime. By the age of 65, anti-C. pneumoniae antibodies are found in 80% of the male and 70% of the female population [2].

Epidemiological studies show an association of serum antibodies against C. pneumoniae and cardiovascular disease [3]. Accumulating data revealed that, due to its obligate intracellular character, C. pneumoniae is able to disseminate via the circulation throughout the body within monocytes and can in this way enter the atherosclerotic lesion [4]. Its ability to infiltrate lesions is illustrated by the observation that in up to 50% of the human atherosclerotic lesions C. pneumoniae is detected [5]. Data from sero-epidemiological and pathology studies underline the possible relation between C. pneumoniae and atherosclerosis, but cannot provide definite proof for a causal relation between C. pneumoniae infection and atherosclerosis. Several clinical trials with antibiotics against these bacteria attempted to demonstrate a causal role in cardiovascular disease, but did not always show decreased pathology [6]. In vitro, C. pneumoniae has various pro-atherosclerotic effects on atheroma-associated cell types. Infection of endothelial cells (ECs) leads to increased expression of adhesion molecules, such as E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) [7], chemokines, such as monocyte chemoattractant protein-1 (MCP-1) [8], and cytokines, such as interleukin (IL)-1, IL-8, and tumor necrosis factor-α (TNF-α) [9], resulting in pro-atherosclerotic processes, such as enhanced adherence and migration of monocytes [8]. Infection of monocytes with C. pneumoniae increases adherence of infected monocytes to ECs [10,11] and promotes low density lipoprotein (LDL) oxidation [12], resulting in accelerated uptake of cholesterol by macrophages and subsequent foam cell formation. Increased LDL oxidation was also observed upon infection of ECs with C. pneumoniae [13]. Smooth muscle cells (SMCs) respond to C. pneumoniae infection by elevated expression of matrix metalloproteinases (MMPs), which may contribute to plaque destabilization [14]. Although in vitro data point to pro-atherosclerotic effects of C. pneumoniae, most of these still have to be confirmed in vivo. Many groups have used animal models to chart the relation between C. pneumoniae infection and cardiovascular disease. However, until now these models did not result in a general consensus about the contribution of C. pneumoniae to atherosclerosis [15,16]. The differences in the observed effects of C. pneumoniae on atherosclerosis may arise from the fact that C. pneumoniae could not always be detected at the site of atherosclerosis after initial lung infection. Therefore, we developed a novel model in which the impact of C. pneumoniae on the arterial wall and subsequent atherogenesis can be studied. C. pneumoniae was delivered to the carotid artery of LDL receptor deficient (LDLr-/-) mice by means of local intra-vascular incubation. Subsequently, the influence of C. pneumoniae, present in the vessel wall, on atherosclerosis was studied by collar placement around the incubated carotid artery. In addition we determined the effect of Mycoplasma pneumoniae on lesion formation in the carotid artery. M. pneumoniae is, like C. pneumoniae, a mild respiratory pathogen that has directly been detected in atherosclerotic lesions [17]. The epidemiological behavior of M. pneumoniae and its ability to produce chronic sequelae following respiratory infection may point to a role for M. pneumoniae in atherosclerosis similar to C. pneumoniae [18]. Since C. pneumoniae may also affect cardiovascular disease by interfering with the thrombogenicity of the vessel wall or atherosclerotic plaque, we also determined the effect of a C. pneumoniae presence in the vascular wall on acute arterial thrombus formation.

1 Methods

1.1 Bacteria

C. pneumoniae strain AR39 (Washington Research Foundation, Washington, USA) was cultured in Buffalo Green Monkey cells. The titer was determined as previously described [19]. M. pneumoniae was kindly provided by Dr Brugman and Dr Thuis from the National Institute for Public Health and Environment (Bilthoven, The Netherlands).

1.2 Delivery of C. pneumoniae and induction of atherosclerosis

Animal work was carried out in compliance with guidelines issued by the Dutch government, which conform to the NIH guidelines. Male LDLr-/- mice, 12–14 weeks old, were put on a “Western-type” diet, containing 0.25% cholesterol and 15% cocoa butter (Special Diet Services, UK). After two weeks mice were anesthetized as previously described [20], and injected intravenously with 200 μl 20 U/ml heparin (Leo Pharma, Netherlands). Subsequently, 15 mice were inoculated with 5 × 105 C. pneumoniae infection forming units (IFU) in 10 μl sucrose–phosphate–glutamic acid (SPG) medium by instillation into the right common carotid artery via the external carotid artery [21]. Carotids of 14 control mice were incubated with a SPG medium. An additional group of mice (n=9) was incubated with 5 × 105 M. pneumoniae bacteria. Suspensions were left inside the common carotid artery for 60 min and subsequently were aspirated from the common carotid artery. Thereafter, the external carotid artery was ligated to prevent blood loss [21]. Immediately after incubation atherosclerosis was induced by bilateral perivascular collar placement [20]. Furthermore, 83 male LDLr-/- mice were locally incubated (40 mice with C. pneumoniae and 43 mice with the SPG medium) without induction of atherosclerosis by collar placement. Twelve mice per time point were sacrificed at day 1, 2, 4, and 7 post-infection for gene expression studies. Another 16 mice were sacrificed 7 days post-infection for DNA isolation or immunohistochemistry and in the remaining 19 animals acute thrombus formation was induced prior to their sacrifice at day 7 post-infection. Acute thrombosis was induced in the right carotid artery by peri-arterial application of FeCl3 (40%) resulting in the formation of platelet and fibrin-rich thrombi, as previously described [22]. A Doppler flow probe was used to measure blood flow. Time to occlusion was defined as the time after initiation of arterial injury with FeCl3, required for blood flow to decline to <0.2 ml/min.

1.3 Immunohistochemistry

For immunohistochemistry carotid arteries were perfused and isolated as previously described [20]. Transverse 5 μm cryosections, prepared in a proximal direction from the carotid bifurcation, were routinely stained with hematoxylin (Sigma Diagnostics, MO) and eosin (Merck Diagnostica, Germany). Each vessel was assessed ∼0.5 mm proximal to the collar, and the site of maximal stenosis was used for morphometric assessment [20]. Corresponding sections were stained immunohistochemically with antibodies against a macrophage-specific antigen (MOMA-2, polyclonal rat IgG2b, diluted 1:50; Research Diagnostics Inc., NJ), alpha smooth muscle cell actin (monoclonal mouse IgG2a (clone 1A4), dilution 1:500; Sigma Diagnostics), and ICAM-1 (monoclonal rat IgG2a (clone BSA2), dilution 1:200; R and D Systems, UK). Slides were incubated with primary antibodies for 2 h at room temperature. As secondary antibodies goat anti-mouse IgG peroxidase conjugate (dilution 1:500; Nordic, The Netherlands) and goat anti-rat IgG alkaline phosphatase conjugate (dilution 1:50; Sigma Diagnostics) were used (one hour of incubation at room temperature), with 3,3′-diamino-benzidine (Sigma Diagnostics), nitro blue tetrazolium (Sigma Diagnostics) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Diagnostics) as enzyme substrates [20]. Sections were stained for collagen by a Picrosirius Red (Direct red 80) staining. Sections were analyzed using a Leica DM-RE microscope and LeicaQwin software (Leica Imaging Systems, UK). Contents of macrophages, smooth muscle cells, and collagen were obtained by dividing the area of specific staining within the intima by the total intima area.

1.4 Detection of C. pneumoniae

DNA was isolated from incubated carotid arteries, after perfusion with PBS, using a QIAamp DNA MiniKit (Westburg, Netherlands). For detection of C. pneumoniae DNA quantitative PCRs were performed on an ABI PRISM 7700 machine (Applied Biosystems, CA) using primers for the outer membrane protein A (OMP A) gene (Table 1), which is highly conserved within C. pneumoniae species [23], and probe 5′-AAACTTAACTGCATGGACCCTTCTTTACTAGG-3′ (FAM), (reverse primers; 300 nM, forward primers; 900 nM, and probes; 200 nM). PCR conditions were the same as used in the gene expression assays. Amounts of C. pneumoniae copies were obtained after performing regression analysis on a standard curve, which was created by performing PCRs under the same conditions on dilutions of plasmids containing the OMP A gene in the presence of representative amounts of DNA isolated from murine carotids.

View this table:
Table 1

Primer sequences used in quantitative PCR

GeneForward primer (5′–′3)Reverse primer (5′–′3)

1.5 Cell culture

Murine macrophages (RAWs) and endothelial cells (H5Vs) were cultured in DMEM containing 10% Fetal Bovine Serum (FBS), 2 mmol/l l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (BioWhittaker Europe) in a humidified atmosphere (5% CO2) at 37 °C. Prior to infection cells were transferred into 24-well plates (2.5 × 105 RAWs/well or 1.0 × 105 H5Vs/well) and incubated overnight.

1.6 In vitro infection

Cells were washed twice with DMEM containing 10% FBS and 2 mmol/l l-glutamine and subsequently inoculated with C. pneumoniae with a m.o.i of 1. Mock infection was performed with a SPG medium. Immediately after inoculation, plates were centrifuged for 60 min with 800 × g at 37 °C. After another 60 min of inoculation with C. pneumoniae, infected cells were washed twice and incubated for 0, 2, 4, 6, and 8 h prior to total RNA isolation.

1.7 Analysis of gene expression by real time quantitative PCR

Total RNA was isolated from RAWs, H5Vs and carotid arteries using TRIzol reagent (Invitrogen, Netherlands) according to manufacturer's instructions. Two arteries were pooled resulting in n=3 per treatment per time point. Generation of cDNA, quantitative RT-PCR, calculations of ratios of target genes and the house hold gene, Hypoxanthine-guanine phosphoribosyl-transferase (HPRT), and the design of primers (Table 1) were performed as previously described [24].

1.8 Data analysis

Values are expressed as mean ± sem. A 2-tailed Student's t-test was used to compare individual groups of mice or cells. Levels of p<0.05 were considered significant.

2 Results

Local incubation of the right common carotid artery with C. pneumoniae did not affect total serum cholesterol levels 5 weeks after collar placement (Fig. 1C). Successful infection of the arterial wall with C. pneumoniae was confirmed by quantitative RT-PCR on the OMP A gene of C. pneumoniae. Every artery incubated with C. pneumoniae was highly positive for the OMP A gene and on average, 6.2 × 104 C. pneumoniae copies were detected in bacteria incubated carotid arteries 7 days post local incubation. C. pneumoniae DNA was absent in lungs, livers and spleens of C. pneumoniae incubated mice and in right carotid arteries of control incubated mice.

Fig. 1

Five weeks after collar placement and simultaneous C. pneumoniae infection mice were sacrificed and carotid arteries were isolated for quantification of lesion formation. Representative cross-sections of collar induced plaques in carotid arteries of LDLr-/- mice are shown after incubation with C. pneumoniae (B) or SPG medium (A). During the experiment total serum cholesterol levels were monitored (C). Using computer assisted morphometric analysis we determined intima area (D), intima/media ratio (E), and intima/lumen ratio (F) after incubation with C. pneumoniae or SPG medium (*=p<0.05).

2.1 Effect of C. pneumoniae presence in the vessel wall on atherosclerosis

After positive identification of the presence of C. pneumoniae we determined the effect of C. pneumoniae on de novo atherosclerosis in carotid arteries of LDLr-/- mice. Five weeks after local incubation of carotid arteries and simultaneous collar placement we determined the degree of atherosclerosis. Incubation of the carotid artery with C. pneumoniae resulted in 1.87-fold increased neointima area (Fig. 1A, B, D; 57,711 ± 10,943 vs. 30,891 ± 9997 μm2), which translated into markedly 2.03-fold increased intima/media ratios (Fig. 1E; 1.27 ± 0.23 vs. 0.62 ± 0.15, p<0.05) and a 1.74-fold increased degree of lumen stenosis (Fig. 1F; 0.53 ± 0.08 vs. 0.30 ± 0.07, p<0.05), as compared to SPG medium incubated arteries. In comparison we performed similar experiments and incubated the carotid artery with M. pneumoniae. Incubation with M. pneumoniae did not affect the extent of atherosclerosis as compared to SPG medium incubated arteries, which was reflected by similar intima area, intima/media and intima/lumen ratios.

2.2 Plaque morphology

Composition of lesions was determined by histological and immunohistochemical staining techniques. The increased lesion size in C. pneumoniae infected arteries was found to be largely attributable to a significant 2.98-fold increased macrophage content of the lesion, as determined by MOMA-2 staining (Fig. 2C; 0.145 ± 0.025 vs. 0.048 ± 0.020, p<0.01). The higher macrophage content was accompanied by a decrease in smooth muscle cells and collagen. In C. pneumoniae infected carotid arteries a tendency towards less staining for alpha-smooth muscle cell actin per intima area was observed as compared with control incubated carotid arteries (Fig. 2F; 0.027 ± 0.007 vs. 0.064 ± 0.031), which was in parallel with the lower collagen content after C. pneumoniae incubation (Fig. 2I; 0.136 ± 0.055 vs. 0.069 ± 0.018). Again incubation with M. pneumoniae did not change the composition of the atherosclerotic plaques as compared to SPG medium incubated carotid arteries.

Fig. 2

Immunohistochemical analysis of plaques developed upon C. pneumoniae incubation (B,E,H) or control incubation (A,D,G). Increased MOMA-2 staining per intima area was observed in bacteria incubated arteries (A,B,C). Both ASMA (D,E) and Picrosirius Red staining (G,H) per intima area were diminished in bacteria incubated arteries as compared to controls (F,I, respectively) (**=p<0.01).

2.3 Inflammatory effect on the vessel wall

Since M. pneumoniae in no way affected the size or composition of the plaque we subsequently focused on the effect of the presence of C. pneumoniae on the expression of inflammatory markers in the vessel wall both at mRNA and protein level in LDLr-/- mice without induction of atherosclerosis. The presence of C. pneumoniae in the vessel wall after local incubation resulted 7 days later in a marked increase in ICAM-1 expression at protein level. Increased ICAM-1 staining was mainly located at the endothelial cell layer (Fig. 3A,B). Furthermore, at day 2 and 4 after local incubation with C. pneumoniae mRNA levels of MCP-1 in C. pneumoniae incubated carotids appeared to be significantly elevated 4.64- (p<0.01) and 6.79-fold (p<0.01), respectively (Fig. 3C), whereas no changes in mRNA levels for ICAM-1, VCAM-1, E-selectin, IL-6, IL-12, and Tissue Factor (TF) were observed.

Fig. 3

Analysis of inflammatory effects of C. pneumoniae on the arterial wall. Seven days post-incubation antibody staining showed that C. pneumoniae in the arterial wall increased ICAM-1 expression (B, arrows) as compared to controls (A). Expression of MCP-1 in the arterial wall on mRNA level was increased markedly at days 2 and 4 after local incubation with C. pneumoniae (▴) as compared with control incubation (□). (C, **=p<0.01).

2.4 Effect of C. pneumoniae on acute arterial thrombosis

The effect of C. pneumoniae in the vessel wall on the thrombogenicity of the carotid artery was assessed using a FeCl3-induced acute arterial thrombosis model. Seven days after infection of the carotid artery with C. pneumoniae no significant differences in time to occlusion after thrombus induction were observed between the SPG medium and the bacteria incubated arteries (1001 ± 138 vs. 1147 ± 164 s, respectively).

2.5 In vitro effect of C. pneumoniae on macrophages and endothelial cells

To further elucidate by which mechanisms C. pneumoniae is capable of stimulating atherogenesis, effects of C. pneumoniae on different cell types, present in the vessel wall, such as ECs and macrophages, were evaluated at mRNA level. The initial level of expression was measured at t=0 h post-infection, which represents the time point immediately after 1 h of centrifugation plus 1 h of incubation with C. pneumoniae. Infection of H5Vs with C. pneumoniae showed an inflammatory response, reflected in increased mRNA levels for MCP-1, for a number of adhesion molecules (E-selectin, ICAM-1, and VCAM-1) and for the cytokines TNF-α and IL-6. Peak values were observed at 2–6 h post-infection (Fig. 4A–F). At 8 h post-infection differences in expression levels between control and infected cells were smaller, while cell viability did not change. In RAWs, TNF-α and MCP-1 expression levels were elevated upon infection (Fig. 4G,H) and reached a maximum within the first hour, which was earlier than in H5Vs.

Fig. 4

Effects of C. pneumoniae on H5Vs (A–F) or RAWs (G,H). Cells were incubated with C. pneumoniae (▴) or SPG medium (□) for 2 h, including 1 h of centrifugation at 800 × g. Subsequently, cells were washed and incubated with medium prior to RNA isolation at indicated timepoints. Quantitative RT-PCR revealed that expression levels of several adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin, were markedly elevated upon infection of H5Vs (A-C). Infection of H5Vs also caused an upregulation of MCP-1, TNF-α and IL-6 (D-F). Expression levels of MCP-1 and TNF-α were also elevated in RAWs upon infection with C. pneumoniae (*=p<0.05, **=p<0.01).

3 Discussion

In the present study, we investigated how direct infection of the vessel wall with C. pneumoniae affects atherosclerosis and thrombogenicity of the carotid artery. Although many groups have studied effects of lung infection with C. pneumoniae on atherosclerosis, little is known about the direct effect of C. pneumoniae in the vessel wall on this inflammatory process. Local administration of C. pneumoniae in coronary arteries of pigs or rabbits has been reported [25,26], but was never performed in atherosclerosis prone mice. Therefore, we established a novel model in which C. pneumoniae is delivered from the luminal side of the carotid artery to the vessel wall. This model mimics the delivery of C. pneumoniae to the arterial wall by infected monocytes and subsequent infection of arterial wall cells by C. pneumoniae after proliferation within macrophages. Using this model we can be sure that the arterial wall is infected. Therefore, this model may be preferred to study the in vivo effect of C. pneumoniae on the arterial wall, since in previous publications effects of C. pneumoniae on atherosclerosis via airway infection were not always accompanied by the positive identification of C. pneumoniae in the arterial wall. The fact that infection via the respiratory tract does not per se lead to positive infection of the arterial wall with C. pneumoniae may explain some of the conflicting results on the effects of C. pneumoniae in atherosclerosis prone mice. Furthermore, this local incubation model offers the opportunity to study the role of C. pneumoniae in the arterial wall without interference of indirect effects, such as effects on lipid metabolism or on systemic inflammation.

By means of local intra-vascular incubation with C. pneumoniae in the common carotid artery it is possible to introduce these bacteria in the vessel wall, which was confirmed by detecting the C. pneumoniae specific Omp A gene in the vessel wall 7 days after incubation in all C. pneumoniae incubated arteries. This detection method circumvents the problems that are met when using immunohistochemistry, in which antigens may be missed while cutting sections or when cross-reactivity with non-chlamydial plaque constituents may occur [27]. In addition, by detecting C. pneumoniae specific DNA it is possible to quantify the number of bacteria that infected the arterial wall. While C. pneumoniae was detected in high amounts in the arterial wall of incubated arteries, these bacteria were not detected in the lungs, liver and spleen of the mice that were locally incubated with C. pneumoniae, indicating the lack of systemic infection upon local incubation. Since ECs form the first barrier that has to be past and C. pneumoniae is able to infect these cells, both in vivo and in vitro, and replicate inside ECs [28,29], entrance of the elementary bodies seems to occur via infection of ECs. Although this model does not entirely mimic the physiological situation, in which bacteria are presumed to enter the vessel wall primarily while residing in monocytes and in which relatively less C. pneumoniae is found in the atherosclerotic plaque, it offers the opportunity to study effects of the actual presence of C. pneumoniae in the vessel wall.

With this local incubation model we demonstrate that C. pneumoniae in the vessel wall enhances collar-induced atherosclerosis in carotid arteries of LDLr-/- mice. Larger lesions were observed in bacteria incubated arteries, which translated into significant larger intima/media and intima/lumen ratios. These results indicate that presence of C. pneumoniae in the arterial wall creates inflammatory stimuli that enhance atherosclerosis and that systemic inflammation as induced by airway infection with C. pneumoniae is not a necessary stimulation for atherosclerosis development. In contrast, infection of the carotid artery with M. pneumoniae, a mild respiratory pathogen that has also been implicated in atherosclerosis, did not affect the size and composition of the atherosclerotic lesions in LDLr-/- mice.

In order to further evaluate the role of C. pneumoniae in different processes involved in atherosclerosis, lesion composition was elucidated by immunohistochemistry. Although larger lesions usually contain relatively less macrophages than smaller lesions, in this experiment the larger lesions in the bacteria incubated group contained relatively more macrophages. Presence of mononuclear infiltrates in vascular tissue upon infection with C. pneumoniae was studied previously. However, the outcome of these studies was not univocal [30–33]. The higher macrophage content observed after C. pneumoniae infection is in line with our in vitro and in vivo data, which showed elevated expression levels of ICAM-1 and MCP-1 upon infection, which can result in increased adherence of circulating monocytes to the endothelium and attraction of these cells into the intima. Furthermore, the finding that C. pneumoniae infection of monocytes can rescue these cells from apoptosis can also contribute to the increased macrophage content [34].

Lesions developed after C. pneumoniae incubation showed a tendency towards decreased α-actin and Picrosirius Red staining, which is indicative for reduced numbers of VSMCs and reduced collagen content, respectively. In vitro studies have shown that proliferation of SMCs can be stimulated by C. pneumoniae both directly and indirectly [35,36]. However, conditioned medium from C. pneumoniae infected SMCs inhibits proliferation of SMCs through induction of prostaglandin E2 [37]. The sum of the in vitro C. pneumoniae induced effects is not well documented in vivo. Distinct observations have been reported in which both stimulatory and inhibiting actions of C. pneumoniae on SMCs proliferation were suggested [25,38,39]. Our study makes a net stimulatory effect of C. pneumoniae on SMCs in vivo less likely. Since collagen is mainly produced by the VSMCs in atherosclerotic lesions, the observed trend in the amount of collagen in the C. pneumoniae incubated carotids fits with the decreased amount of SMCs observed in the atherosclerotic lesions. Several groups have reported that upon infection with C. pneumoniae both macrophages and SMCs produce MMPs [40,41], which are able to degrade collagen. Previously, enhanced MMP-2 and MMP-9 production was observed in atherosclerotic lesions upon infection with C. pneumoniae in LDLr/ApoE double knock out mice [39]. This lesion morphology with more macrophages and the lack of compensatory elevated amounts of VSMCs and collagen point to a net destabilizing effect of C. pneumoniae.

Presence of C. pneumoniae in the vessel wall resulted in enhanced expression of MCP-1 at mRNA level. It remained unclear from these experiments to which cell types this increased expression can be contributed. However, since ECs, macrophages and VSMCs [41] in vitro show elevated expression of MCP-1 upon infection with C. pneumoniae they can be all involved in this response. Immunohistochemistry revealed increased ICAM-1 protein expression mainly on ECs, which, combined with the found elevated MCP-1 expression level, could contribute to the increased macrophage content, as observed in infected lesions. The lack of increased expression of ICAM-1 at mRNA level could be explained by the fact that expression levels at mRNA level were not specifically measured in the endothelium, but in the total vessel wall, which may dilute the ICAM-1 signal.

In vitro studies have shown that C. pneumoniae increases the expression of the pro-coagulant proteins TF and plasminogen activator inhibitor-1 in ECs, SMCs and macrophages [42,43], and accelerates clotting and phosphatidylserine exposure in several cell types [44]. However, in our in vivo model, in which acute thrombus formation was induced by peri-arterial application of FeCl3, no pro-thrombotic effect was observed 7 days after delivery of C. pneumoniae to the arterial wall. In addition, no upregulation of mRNA levels for TF was detected in the vessel wall during the first week after delivery of C. pneumoniae. These data suggest that previously shown in vitro indications for increased thrombogenicity upon infection with C. pneumoniae may be compensated by antithrombotic actions of C. pneumoniae in vivo, such as induction of the anti-thrombotic cytokine IL-10 [34,45], resulting in the absence of a significant effect of C. pneumoniae in the vessel wall on thrombogenicity.

In vitro infections of mouse ECs and macrophages with C. pneumoniae were performed to support the results observed in vivo. As demonstrated previously, infection of ECs induced an inflammatory response marked by elevated mRNA levels of IL-6, TNF-α, E-selectin, ICAM-1, and VCAM-1. Combined with the elevated MCP-1 expression in both macrophages and ECs, these data suggest that C. pneumoniae promotes monocyte adhesion and migration to the inner layers of the vessel wall, which is in accordance with the in vivo results.

In conclusion, this study demonstrates that direct infection of the carotid artery with C. pneumoniae enhances atherosclerosis in a novel model. C. pneumoniae affects the composition of the developing lesions towards a more vulnerable morphology via an increase in macrophage content, which was not observed after infection of the carotid artery with M. pneumoniae. Therefore, this study supports the specific contribution of C. pneumoniae to the progression of atherosclerosis. Enhanced macrophage content caused the major increase in lesion formation, and chemokines contributed to this increase. This local delivery model offers the opportunity to further elucidate the site-specific influences of C. pneumoniae on atherosclerosis.


This study was supported by grant 2000.198 and grant 2000D040 from the Netherlands Heart Foundation. J. Kuiper is the Established Investigator of the Netherlands Heart Foundation (grant 2000D040).


  • Time for primary review 36 days


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