Chlamydophila pneumoniae Infection Leads to Smooth Muscle Cell Proliferation and Thickening in the Coronary Artery without Contributions from a Host Immune Response
2009; Elsevier BV; Volume: 176; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2010.090645
ISSN1525-2191
AutoresJustin Deniset, Paul Cheung, Elena Dibrov, Kaitlin Lee, Sarah N. Steigerwald, Grant N. Pierce,
Tópico(s)Reproductive Physiology in Livestock
ResumoChlamydophila pneumonia (C. pneumonia) infection has been associated with the progression of atherosclerosis. It remains unclear, however, whether C. pneumoniae in the absence of an immune response can alone initiate atherogenic events within a complex vessel environment. Left anterior descending coronary arteries isolated from porcine hearts were dissected and placed in culture medium for 72 hours before infection with C. pneumoniae. C. pneumoniae replicated within the arterial wall for the duration of the experiment (up to 10 days). A significant increase in chlamydial-HSP60 protein expression from day 2 to 10 post-infection (pi) indicated the presence of metabolically active C. pneumonia within infected vessels. Significant arterial thickening in infected coronary segments was observed by a considerable decrease in the ratio of lumen to total vessel area (48 ± 3% at day 4 pi versus 23 ± 3% at day 10 pi) and a significant increase in the ratio of media to luminal area (113 ± 16% at day 4 pi versus 365 ± 65% at day 10 pi). Structural changes were accompanied by an up-regulation of host HSP60 and proliferating cell nuclear antigen expression levels. Immunohistochemical staining confirmed proliferating cell nuclear antigen expression to be primarily localized within smooth muscle cells of the medial area. These results demonstrate that C. pneumoniae infection can stimulate arterial thickening in a complex vessel environment without the presence of a host immune response and further supports the involvement of HSP60 in this action. Chlamydophila pneumonia (C. pneumonia) infection has been associated with the progression of atherosclerosis. It remains unclear, however, whether C. pneumoniae in the absence of an immune response can alone initiate atherogenic events within a complex vessel environment. Left anterior descending coronary arteries isolated from porcine hearts were dissected and placed in culture medium for 72 hours before infection with C. pneumoniae. C. pneumoniae replicated within the arterial wall for the duration of the experiment (up to 10 days). A significant increase in chlamydial-HSP60 protein expression from day 2 to 10 post-infection (pi) indicated the presence of metabolically active C. pneumonia within infected vessels. Significant arterial thickening in infected coronary segments was observed by a considerable decrease in the ratio of lumen to total vessel area (48 ± 3% at day 4 pi versus 23 ± 3% at day 10 pi) and a significant increase in the ratio of media to luminal area (113 ± 16% at day 4 pi versus 365 ± 65% at day 10 pi). Structural changes were accompanied by an up-regulation of host HSP60 and proliferating cell nuclear antigen expression levels. Immunohistochemical staining confirmed proliferating cell nuclear antigen expression to be primarily localized within smooth muscle cells of the medial area. These results demonstrate that C. pneumoniae infection can stimulate arterial thickening in a complex vessel environment without the presence of a host immune response and further supports the involvement of HSP60 in this action. Chlamydophila pneumoniae (C. pneumoniae) is an obligate intracellular parasite that causes respiratory illness in humans. 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The study of C. pneumoniae-induced atherogenesis on any in vivo model is inevitably complicated by the contributions of host-immune and inflammatory responses (T-cells and cytokines), which are thought to be strong contributors to the lesions. C. pneumoniae infection can augment the secretion of inflammatory markers by endothelial cells.25Coombes BK Mahony JB Chlamydia pneumoniae infection of human endothelial cells induces proliferation of smooth muscle cells via an endothelial cell-derived soluble factor (s).Infect Immun. 1999; 67: 2909-2915PubMed Google Scholar, 26Coombes BK Chiu B Fong IW Mahony JB Chlamydia pneumoniae infection of endothelial cells induces transcriptional activation of platelet-derived growth factor-B: a potential link to intimal thickening in a rabbit model of atherosclerosis.J Infect Dis. 2002; 185: 1621-1630Crossref PubMed Scopus (30) Google Scholar, 27Hirono S Dibrov E Hurtado C Kostenuk A Ducas R Pierce GN Chlamydia pneumoniae stimulates proliferation of vascular smooth muscle cells through induction of endogenous heat shock protein 60.Circ Res. 2003; 93: 710-716Crossref PubMed Scopus (68) Google Scholar The up-regulation of HSP60 in human atheroma and the corresponding elevated levels HSP60 antibodies can also lead to autoimmune injury in the arterial environment.29Huittinen T Leinonen M Tenkanen L Manttari M Virkkunen H Pitkanen T Wahlstrom E Palosuo T Manninen V Saikku P Autoimmunity to human heat shock protein 60, chlamydia pneumoniae infection, and inflammation in predicting coronary risk.Arterioscler Thromb Vasc Biol. 2002; 22: 431-437Crossref PubMed Scopus (95) Google Scholar, 34Kol A Sukhova GK Lichtman AH Libby P Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression.Circulation. 1998; 98: 300-307Crossref PubMed Scopus (502) Google Scholar, 35Kol A Bourcier T Lichtman AH Libby P Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages.J Clin Invest. 1999; 103: 571-577Crossref PubMed Scopus (464) Google Scholar, 36Schett G Xu Q Amberger A Van der Zee R Recheis H Willeit J Wick G Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity.J Clin Invest. 1995; 96: 2569-2577Crossref PubMed Scopus (255) Google Scholar, 37Chen W Syldath U Bellmann K Burkart V Kolb H Human 60-kDa heat-shock protein: a danger signal to the innate immune system.J Immunol. 1999; 162: 3212-3219PubMed Google Scholar However, the question of whether C. pneumoniae infection directly injures coronary arteries and induces thickening without the effects of the immune system remains unanswered. It is difficult to answer this question in an in vivo environment because the immune system is fully activated by the infectious agent. Data from in vitro studies are not ideal for studying C. pneumoniae induced proliferation and thickening because cell lines are naturally proliferative and lack the structural complexity of a vessel. In the current study, we used a novel ex vivo organ culture model that allowed us to study the direct consequences of the C. pneumoniae infection in the complex arterial environment without the confounding contributions of a host immune system. Using this novel approach, we observed C. pneumoniae replication and the spread of the infection into the medial layer of coronary arteries. Furthermore, C. pneumoniae infection induced arterial wall thickening and expression of proliferation and stress markers. This is the first report that shows that C. pneumoniae infection can stimulate an atherogenic response independently of the host immune reactions. Cycloheximide, bradykinin acetate, 9,11-Dideoxy-11α, 9α-epoxy-methanoprostaglandin F2α (u46619), sodium nitroprusside, Hoescht, anti–proliferating cell nuclear antigen (PCNA) antibody, and anti-smooth muscle actin antibody were obtained from Sigma-Aldrich (St-Louis, MO). The anti–Chlamydia genus antibody was obtained from Argene (Verniolle, France). The anti-PCNA antibody was obtained from Bethyl Laboratories Inc. (Montgomery, TX). The anti-chlamydial heat-shock protein 60 antibody was obtained from Affinity BioReagents (Golden, CO). The anti-mammalian heat-shock protein 60 was obtained from Assay Designs-Stressgen (Ann Arbor, MI). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Abcam (Cambridge, MA). The HRP conjugated anti-mouse IgG was obtained from Millipore (Billerica, MA). The Alexa-488–conjugated anti-mouse IgG and Alexa-488-conjugated anti-rabbit IgG antibodies were obtained from Invitrogen (Carlsbad, CA). The Texas Red-conjugated anti-mouse IgG antibody was obtained from Jackson Laboratory (Bar Harbor, ME). C. pneumoniae AR39 strain was obtained from the University of Washington, Seattle, WA. The organism was propagated in HL cells as described previously.38Kuo CC Grayston JT A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR.J Infect Dis. 1990; 162: 755-758Crossref PubMed Scopus (113) Google Scholar The purified organism was resuspended in chlamydial sucrose-phosphate-glutamate medium and stored at −80°C until use. The titer of C. pneumoniae was determined in cycloheximide-treated HL cells, and concentrations used were expressed as inclusion-forming units per ml.39Furness G Graham DM Reeve P The titration of trachoma and inclusion blennorrhoea viruses in cell cultures.J Gen Microbiol. 1960; 23: 613-619Crossref PubMed Google Scholar The coronary artery explant and organ culture method has been adapted from Saward et al.40Saward L Zahradka P Coronary artery smooth muscle in culture: migration of heterogeneous cell populations from vessel wall.Mol Cell Biochem. 1997; 176: 53-59Crossref PubMed Scopus (53) Google Scholar Whole hearts from 10-month-old neutered male swine were obtained from a local abattoir. The left anterior descending coronary artery was flushed with standard phosphate saline buffer (PBS) supplemented with an antibiotic mixture (penicillin G, 1500 units/ml; streptomycin sulfate, 1500 μg/ml; amphotericin B, 2.5 μg/ml; Invitrogen) and subsequently dissected out of the heart and cleaned of adhering fat and connective tissue. The explanted artery was cut into segments of 5 mm in length and incubated at 37°C and 5% CO2 in organ culture medium (Dulbecco's Modified Eagle's Medium; Invitrogen, supplemented with 20% fetal bovine serum; Hyclone) supplemented with antibiotics (penicillin G, 1050 units/ml; streptomycin sulfate, 1050 μg/ml; amphotericin B, 2.5 μg/ml; Invitrogen). Every 24 hours, explants were placed in fresh organ culture medium with gradually decreasing amount of antibiotics. At 72 hours, the coronary segments were washed with PBS and incubated in the organ culture medium for 3 hours. The segments were then infected with C. pneumoniae. No antibiotics were used for the duration of the experiment. To perform the infection, each coronary segment was placed into an isolated well of a 96-well culture plate. The segments were oriented upright within the well so that C. pneumoniae (5 × 106 inclusion-forming units in 100 μl) could be applied directly into the lumen of each coronary vessel. After three hours of incubation at 37°C and 5% CO2, each coronary segment was transferred to an isolated well of a 24-well plate containing 1.5 ml of organ culture medium. The vessels were incubated for a maximum of 10 days. The incubation medium was changed every 48 hours. Coronary segments were collected for analysis immediately after infection and from day 2 to day 10 pi. Heat-inactivated C. pneumoniae (mock-infection) was used for controls in each experiment. Infected and mock-infected coronary segments were collected at various time points pi: day 2, day 4, day 6, day 8, and day 10. The segments were fixed in ethanol, rehydrated, and subsequently equilibrated in 30% sucrose solution before mounting in OCT compound (Tissue-tek). These OCT-mounted segments were slowly frozen to −80°C, cut (HM 500 OM Cryostat, Microm) into 6-μm sections, and placed on positively coated glass slides (Fisher). OCT was washed off the glass slides with PBS, and antigen retrieval was performed with the Unmasking solution (Vector Laboratories) at 65°C for 25 minutes. To detect C. pneumoniae in the specimen, anti–Chlamydia genus antibody (1:100) was applied according to manufacturer's protocol. Alexa-488–conjugated anti-mouse IgG (1:800) was used as a secondary antibody. To localize PCNA expression within the vessel, anti-smooth muscle actin (1:500) and anti-PCNA (1:100) were used. Texas Red–conjugated anti-mouse IgG (1:1000) and Alexa-488–conjugated anti-rabbit IgG (1:1000) were used as secondary antibodies for the respective primary antibodies. Hoescht staining solution (5 ng/ml) was added to the slides to identify nuclei. Slides were then fixed with FluoroSave reagent (Calbiochem) to preserve fluorescence. The location and number of C. pneumoniae inclusion bodies (IB) in the coronary cross-sections were visualized as green fluorescent signals at 40× magnification on an inverted microscope (Nikon, TE-2000s). In each field of view, the number of fluorescent IBs was calculated using imaging software (Adobe photoshop CS3 extended). To identify apoptotic cells within the vessel, terminal dUTP nick-end labeling (TUNEL) assays using the FragEL DNA fragmentation detection kit (Calbiochem) were performed on coronary cross-sections. Cross-sections pretreated with DNase I were used as positive controls. To determine the extent of media thickening, coronary segments were collected at four time points pi: day 4, day 6, day 8, and day 10. The extent of media thickening in C. pneumoniae–infected segments was compared with corresponding mock-infected control segments. The segments were fixed and mounted as described above before being cut into 6-μm cross-sections. Hematoxylin and eosin (H/E) staining as well as elastic staining were applied to these cross-sections. Images of the cross-sections were obtained at 4× magnification (Nikon TE-2000s). Imaging software (Adobe photoshop CS3 extended) was used to quantify the pixel area of the lumen, media, and total vessel for each coronary cross section. To assess protein expression levels, coronary segments were collected at four time points: day 0 (before infection), days 2 to 4, days 5 to 6, and days 8 to 10 pi. These collection intervals were chosen to coincide with the C. pneumoniae replication cycle. Three independent experiments were performed. In each experiment, two coronary segments per time point per treatment were collected, rinsed with PBS, flash-frozen in liquid nitrogen and subsequently stored at −80°C. Paired coronary segments were ground and resuspended in RIPA buffer (50 mmol/L TrisHCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton 100, 0.1% SDS, 0.5% Na Deoxycholate, 1 μg/ml Leupeptin, 1 mmol/L PMSF, 1 mmol/L protease inhibitor cocktail, 1 mmol/L DTT and 1 mmol/L Benzimidine). Proteins were separated using a 10% denaturing polyacrylamide gel and transferred electrophoretically onto a nitrocellulose membrane. Membranes were incubated with anti-chlamydial heat-shock protein 60, anti-mammalian heat-shock protein 60, and anti-PCNA antibodies. HRP conjugated anti-mouse IgG was used as a secondary antibody. The level of GAPDH was used as a loading control. Bands were visualized with the Supersignal West Pico Chemiluminescent Substrate (Pierce) and subsequently quantified by densitometry (Software: Quantity One, Bio-Rad). To determine tissue viability over the duration of the experiment, untreated coronary segments were collected at days 0, 5, and 10 pi to coincide with the start, mid-point, and end of the infection timeline. These tissues were used for vascular function experiments. The 5-mm coronary rings were fastened in an organ bath with surgical wire, perfused with Krebs-Henseleit solution, aerated with 95% O2 and 5% CO2, and equilibrated at 37C and pH 7.4. Vascular function was measured with a force transducer (FSG-01/20, Experimentia Ltd, Budapest, Hungary) as mechanograms of tension. The coronary segments were brought to a basal tension of 5 g and then contracted three times with 47 mmol/L KCl, with washout periods using Krebs solution between each contraction. To assess relaxation responses, rings were precontracted with 30 nmol/L u46619 and allowed to reach a steady state of contraction. Bradykinin was then administered without washout at concentrations of 10−10 to 10−6 mol/L to develop an endothelial-dependent response curve. After washout and a second precontraction with 30 nmol/L u46619, sodium nitroprusside was administered at concentrations of 10−7 to 10−4 to develop an endothelial independent response curve. After a washout, a dose–response curve to u46619 was constructed with concentrations of 0.3 to 300 nmol/L. Data are presented as mean ± SEM unless otherwise stated. Differences in C. pneumoniae infected and mock C. pneumoniae infected data including the levels of protein expression, the ratio of lumen area to vessel area, the ratio of media to lumen, and the number of inclusion bodies in coronary cross-sections were tested by one-way analysis of variance followed by a Duncan's post hoc test. A probability of P < 0.05 was considered statistically significant. Vascular function was determined in untreated coronary vessels at days 0, 5, and 10 pi. There were no significant changes in maximal contractile force development to KCl or to u46619 at any of the day points (Figure 1, A and B). Furthermore, both endothelial-dependent and -independent relaxation response curves to bradykinin and sodium nitroprusside, respectively, were maintained for the duration of the experiment (Figure 1, C and D). Chlamydial IBs are represented as green fluorescent signals within the coronary cross-sections (Figure 2). Magnified views of both the endothelial layer (bottom right corner) and the medial layer (top left corner) are provided within each figure panel for improved visualization of inclusion body distribution. C. pneumonia infected tissues displayed the presence of IBs at all time points, with variations in their distribution, number, and intensity. On day 2 pi, intense green fluorescence was localized in the endothelial layer but not in the smooth muscle cells (Figure 2A). On day-4 pi, C. pneumoniae IBs were still detected in the endothelium but were now also visible in the smooth muscle layer (Figure 2B). This distribution trend continued through days 6, 8, and 10 pi. At these time points, IBs were primarily localized in the media and only occasionally in the endothelium (Figures 2, C, D, E, and F). The number of IBs were quantified (Figure 3A), with the exception of IBs at day-2 pi, when it was too difficult to resolve the continuum of green fluorescence in the endothelial layer into individual IB units. From days 4 to 10 pi, a significantly (P < 0.0003) increasing number of IB was observed. C. pneumoniae IBs at days 8 and 10 pi were also noticeably larger in size than IBs detected at earlier day points. There were no C. pneumoniae IBs detected in mock-infected controls.Figure 3C. pneumoniae replication and metabolic activity within isolated coronary sections. A: The number of C. pneumoniae inclusion bodies at days 4 to 10 pi (mean ± SEM, n = 3, *P < 0.001). B: Western blot analysis of chlamydial HSP60 (c-HSP60) expression in the coronary segments over the course of infection. Representative Western blots of c-HSP60 and GAPDH (loading control) are displayed above the graph. Normalized protein expression levels were represented as fold increase of the basal level. Basal level of c-HSP6
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