Chlamydia pneumoniae Decreases Smooth Muscle Cell Proliferation through Induction of Prostaglandin E₂ Synthesis

Atherosclerotic diseases have been linked to Chlamydia pneumoniae infection (reviewed in reference 1). The organism has been found within endothelial cells, macrophages, and smooth muscle cells (SMC) in atherosclerotic plaques (25). C. pneumoniae may contribute to the pathogenesis of atherosclerosis by eliciting cellular responses associated with inflammation, cell proliferation, and tissue remodeling (13).

During atherogenesis, SMC migrate from the media to the intima. Proliferation of neointimal SMC and production of extracellular matrix proteins by these cells result in the formation of a fibrous cap overlying the prothrombotic lipid-rich core of the atherosclerotic plaque (11). Growth factors, such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), are thought to be largely responsible for the accumulation of SMC in the intima (8, 10, 14). Coombes et al. reported that C. pneumoniae infection of endothelial cells increases the expression of PDGF-BB (PDGF containing two B chains), which may contribute to the intimal thickening of aortic tissue in Chlamydia-infected rabbits (4). Besides endothelial cells, SMC themselves are discussed as an important source of growth factors in atherosclerotic plaques (14). A previous paper reported that SMC produce increased amounts of bFGF in response to infection with C. pneumoniae (18). Therefore, the aim of this study was to investigate the effects of conditioned medium from Chlamydia-infected SMC on the proliferation of uninfected SMC.

Human vascular SMC (C-12511; PromoCell, Heidelberg, Germany) were infected with C. pneumoniae TW-183 (obtained from the Institute of Ophthalmology, London, United Kingdom) at a multiplicity of infection (MOI) of 5 as previously described (17). For heat inactivation, chlamydial suspensions were held at 75°C for 10 min prior to inoculation onto cell monolayers. For UV inactivation, chlamydial suspensions were placed under a UV lamp (15 W at 30 cm) for 15 min. Mock-infected and infected SMC were incubated with SMC basal medium (PromoCell) containing 1% fetal calf serum (FCS) but no antibiotics. In some experiments, mock-infected and infected SMC were treated with NS-398 {N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide; Qbiogene-Alexis, Grünberg, Germany}. Chlamydial inclusions in infected cultures were visualized by immunofluorescence staining as previously described (17). Conditioned media from infected and mock-infected cultures were collected at 2, 24, 48, and 72 h after infection and clarified by centrifugation at 20,000 × g for 30 min before inoculation onto uninfected SMC.

DNA synthesis in SMC was assessed as described by Padró et al. with modifications (15). SMC were seeded onto 96-well plates at a density of 10³ cells per well and incubated with basal medium containing 10% FCS for 24 h. The cells were then maintained in basal medium containing 1% FCS for a further 72 h. After this period, cells were incubated with conditioned media from infected and mock-infected SMC (100 μl per well). In some experiments, SMC were treated with medium containing 1% FCS and prostaglandin E₂ (PGE₂; Qbiogene-Alexis). Complete SMC growth medium consisting of basal medium, 5% FCS, 0.5 ng of human recombinant epidermal growth factor (PromoCell) per ml, 2 ng of human recombinant bFGF (PromoCell) per ml, and 5 μg of bovine insulin (PromoCell) per ml was used as a positive control for SMC proliferation. The next day, 5-bromo-2-deoxyuridine (BrdU; 10 μM; Amersham Biosciences, Freiburg, Germany) was added, and the cultures were maintained for a further 24 h. Incorporation of BrdU into the DNA of proliferating cells was measured by enzyme-linked immunosorbent assay (Amersham Biosciences) according to the manufacturer's protocol. Results were expressed in percentages as the ratio of absorbance of conditioned medium to that of SMC growth medium.

For counting cells, SMC were plated onto 12-well plates at 10⁴ cells per well, incubated with medium containing 10% FCS for 24 h, and then maintained in medium with 1% FCS for 72 h. The next day, SMC were treated with conditioned medium (1 ml per well) for a period of 4 days. For some experiments, cells were overlaid with 1:1 mixtures of complete SMC growth medium and conditioned medium. SMC numbers were determined every other day. Cells were trypsinized, resuspended in medium, and counted under the microscope in a Neubauer chamber.

Levels of PGE₂ in conditioned medium were measured by enzyme-linked immunosorbent assay (R&D Systems, Wiesbaden, Germany) according to the manufacturer's protocol.

To examine whether soluble factors produced by C. pneumoniae-infected SMC modulate SMC proliferation, conditioned media collected from mock-infected and infected SMC cultures were examined for the ability to stimulate DNA synthesis in SMC. When SMC were incubated with medium containing 1% FCS as used in the infection experiments, the incorporation of BrdU was decreased by 60% compared to that measured when SMC were incubated with complete SMC growth medium (Fig. 1A). We found no differences in levels of DNA synthesis between SMC cultured with fresh medium containing 1% FCS and SMC stimulated with conditioned medium from mock-infected cells. However, conditioned medium harvested from Chlamydia-infected cells at 24, 48, and 72 h after infection caused a significant reduction in BrdU uptake in SMC compared to that in SMC incubated with conditioned medium from mock-infected SMC (Fig. 1A). Conditioned medium collected at 2 h after infection had no inhibitory effect on BrdU incorporation (Fig. 1A). The findings suggest that infected SMC released factors inhibiting SMC proliferation. The induction of antimitogenic factors required the infection of SMC by viable chlamydiae because conditioned medium prepared after exposure of cells to heat- and UV-inactivated bacteria did not markedly decrease BrdU uptake in SMC (Fig. 1B). Antimitogenic activities of conditioned medium could be found following chlamydial infection of SMC at different MOIs ranging from 2 to 20 (data not shown).

PGE₂ has been described to function as a negative regulator of SMC proliferation (24). C. pneumoniae stimulated the production of PGE₂ by SMC (Fig. 2A). The release of PGE₂ was slightly increased at 2 h, and large amounts of PGE₂ were produced at 24, 48, and 72 h after infection (Fig. 2A). Because cyclooxygenase-2 (COX-2) is one of the key enzymes catalyzing PGE₂ synthesis, conditioned medium was prepared in the presence of NS-398, a selective inhibitor of COX-2 activity (7). Treatment of infected cells with NS-398 not only suppressed the production of PGE₂ but also abolished the capacity of conditioned medium to inhibit DNA synthesis in SMC, indicating that the antiproliferative factor induced by C. pneumoniae was a prostanoid (Fig. 2B and C). The percentages of Chlamydia-positive cells in infected cultures did not significantly differ between SMC with NS-398 treatment and those without NS-398 treatment (9% ± 2% versus 12% ± 3% inclusion-positive cells following infection at an MOI of 5). In comparative experiments, exogenous PGE₂ induced a significant decrease in BrdU uptake in SMC at a concentration of 3,000 pg per ml, which corresponds to the amounts of PGE₂ found in conditioned medium harvested from Chlamydia-infected cells at 24, 48, and 72 h after infection (Fig. 2D). In contrast, a low PGE₂ concentration of 300 pg per ml had no inhibitory effect on BrdU incorporation (Fig. 2D).

Direct cell counting was performed to evaluate whether the inhibition of DNA synthesis in SMC after exposure to conditioned medium from Chlamydia-infected cells was accompanied by decreased cell proliferation. Conditioned medium from mock-infected cells had only moderate proliferative effects on SMC compared to complete SMC growth medium (Fig. 3A). However, cell numbers after 4 days of incubation were significantly higher in cultures overlaid with conditioned medium from mock-infected cells than in cultures treated with conditioned medium from C. pneumoniae-infected SMC (Fig. 3A). In further experiments, we investigated whether conditioned medium from Chlamydia-infected SMC can decrease the proliferation of SMC in the presence of complete growth medium. When conditioned medium from infected cells was added to SMC growth medium at a ratio of 1:1, cell numbers after 4 days in culture were reduced by 50% compared to those in cultures treated with conditioned medium from mock-infected SMC or fresh medium containing 1% FCS (Fig. 3B).

Arterial C. pneumoniae infection may contribute to the pathogenesis of atherosclerosis by modulating SMC proliferation. This study shows that the infection of SMC by C. pneumoniae induces antiproliferative effects on uninfected SMC via secretion of PGE₂.

C. pneumoniae infection of SMC stimulated the production of PGE₂, which could be abolished by treating the cells with the COX-2 inhibitor NS-398. This observation corresponds to those from previous work in which the induction of COX-2 expression and PGE₂ synthesis in epithelial cells upon chlamydial infection was demonstrated (9, 26). PGE₂ is known to inhibit mitogenesis in SMC by elevating cyclic AMP levels (16, 24). The suppression of PGE₂ synthesis in SMC by NS-398 treatment abolished the capacity of conditioned medium to decrease SMC proliferation. Furthermore, the stimulation of SMC with exogenous PGE₂ at a concentration found in conditioned medium caused a significant reduction in DNA synthesis. PGE₂ has been described as being more effective in inhibition of SMC growth than other prostaglandins (12, 23). However, it cannot be excluded that other COX-2 products may also contribute to the antiproliferative effects of conditioned medium.

The results of our proliferation assays seem to be in contrast to those of other studies that investigated effects of C. pneumoniae infection on SMC proliferation. Coombes et al. described an intimal thickening of aortic tissue in rabbits following infection with C. pneumoniae (4). The intimal thickening of the aortas correlated with the detection of both PDGF-BB and chlamydial antigen (4). The same group also reported that the conditioned medium from Chlamydia-infected endothelial cells stimulates the growth of SMC (3). The factor responsible for these effects was not identified, although increased expression of PDGF-BB by infected endothelial cells may be involved (4). It has previously been shown that C. pneumoniae stimulates SMC to produce bFGF (18). However, the findings of the present study indicate a positive net balance of growth inhibitory PGE₂ versus potential growth stimulatory factors in the conditioned medium of infected SMC. In atherosclerotic plaques, PGE₂ released from Chlamydia-infected SMC may inhibit the proliferation of neighboring SMC. On the other hand, it has to be considered that C. pneumoniae may also directly affect SMC growth (19, 20). Sasu et al. reported that chlamydial heat shock protein 60 can activate the proliferation of SMC via Toll-like receptor 4 (19).

The proliferation of SMC in the fibrous cap of atherosclerotic plaques is accompanied by the deposition of extracellular matrix proteins, thereby promoting plaque stability (11). Acute ischemic syndromes are usually due to the rupture of instable atherosclerotic plaques and subsequent thrombosis (11, 21). In symptomatic lesions, the fibrous cap can rupture as a result of matrix degradation caused by matrix metalloproteinases and the reduction in the number of SMC following increased apoptosis and decreased proliferation (11, 21, 22). Since COX-2 and PGE₂ synthase are overexpressed in symptomatic lesions, the up-regulation of PGE₂ is discussed as playing a role in decreased SMC proliferation and in plaque destabilization (2, 5). Recently, Ezzahiri et al. reported that the infection of low-density lipoprotein receptor/ApoE^−/− mice with C. pneumoniae results in a significant decrease in the SMC content of the fibrous cap of atherosclerotic lesions in the aortic arch (6). The finding that C. pneumoniae infection of SMC decreases SMC proliferation via stimulation of PGE₂ synthesis suggests that the interaction of vascular SMC with C. pneumoniae may contribute to mechanisms promoting the destabilization of atherosclerotic plaques.