Published online before print January 31, 2008, doi: 10.1161/ATVBAHA.107.156000
© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Role for Staphylococci in Misguided Thrombus Resolution of Chronic Thromboembolic Pulmonary Hypertension
From the Departments of Cardiology (D.B., J.J., B.R., M.-K.R., H.P., C.A., I.M.L.), Biomedical Research (H.B.), Infectious Diseases and Chemotherapy (A.G.), and Cardiothoracic Surgery (W.K.) of the Medical University of Vienna, and the Medical Department V (M.K.) of the Wilhelminenspital der Stadt Wien, Austria.
Correspondence to Irene M Lang, MD, Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. E-mail irene.lang{at}meduniwien.ac.at
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Abstract |
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Objective— Acute pulmonary emboli usually resolve within 6 months. However, in 0.1% to 3.8% of cases thrombus transforms into fibrous masses. If vascular obstruction is severe, the resulting condition is chronic thromboembolic pulmonary hypertension (CTEPH). Patients who carry ventriculo-atrial (VA-) shunts for the treatment of hydrocephalus and report a history of shunt infection are at an increased risk for CTEPH. Because CTEPH lacks traditional plasmatic risk factors for venous thromboembolism, we hypothesized that delayed thrombus resolution rather than abnormal coagulation is important, and that bacterial infection would be important for this misguidance.
Methods and Results— Human CTEPH thromboemboli were harvested during pulmonary endarterectomy. The effects of Staphylococcal infection on thrombus organization were examined in a murine model of stagnant-flow venous thrombosis. Staphylococcal DNA, but not RNA, was detected in 6 of 7 thrombi from VA shunt carriers. In the mouse model, staphylococcal infection delayed thrombus resolution in parallel with upregulation of transforming growth factor (TGF) beta and connective tissue growth factor.
Conclusions— In the present work, we propose a mechanism of disease demonstrating that infection with Staphylococci enhances fibrotic vascular remodeling after thrombosis, resulting in misguided thrombus resolution. Thrombus infection appears to be a trigger in the evolution of CTEPH.
Mechanisms underlying thrombus persistence in chronic thromboembolic pulmonary hypertension (CTEPH) are unknown. In the present work, we analyzed human surgical CTEPH specimens and experimental murine venous thrombi. Our findings suggest a pivotal role for bacterial infection in the fibrotic organization process and persistence of thrombotic material in CTEPH.
Key Words: pulmonary embolism • thrombus resolution • infection
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Introduction |
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The natural history of acute pulmonary thromboemboli is to undergo almost complete resolution within 6 months.1 However, in 0.1% to 3.8% of survived acute events,2,3 thromboemboli undergo an organization process leading to permanent fibrotic obstruction of the pulmonary vascular bed. Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by predominantly major-vessel obstructions4 resulting in increased pulmonary vascular resistance. The molecular mechanisms underlying thrombus persistence are unknown.5
CTEPH is largely understood as of thromboembolic origin. However, patients with CTEPH lack classic plasmatic thromboembolic risk factors,6 and systemic7 or local8 imbalances of fibrinolytic proteins in the pulmonary arterial wall. In addition, it is impossible to induce the disease in animal models by repeated embolizations,9 suggesting alternative nonthromboembolic hypotheses.10 Increased plasminogen activator inhibitor (PAI)-111 and von Willebrand factor (vWF) (unpublished data, Bonderman and Lang, 2003) expressions on endothelial cells of the thrombus surface support the concept of a localized abnormal prothrombotic gene expression pattern furthering in situ thrombosis.
We have recently observed that patients who carry ventriculo-atrial (VA)- shunts for the treatment of hydrocephalus, or pacemaker leads and have a history of shunt/lead infection are at an increased risk for CTEPH12 and carry an unfavorable prognosis.13 Shunt infection is common in patients with VA-shunts, and Staphylococcus (S) aureus or epidermidis is responsible for up to one-half of these infections,14 leading to thrombosis and device failure.15 An association between tissue fibrosis and infection has been described in a variety of tissues, such as liver,16 lung,17 and intestine,18 and bacterial pathogens are drivers of unrestrained wound healing.19 Bacterial infection induces upregulation of TGF beta (TGFβ),20 a profibrotic cytokine, and connective tissue growth factor (CTGF),18 a matricellular protein that regulates collagen synthesis and maintains fibrosis.21,22 We hypothesized that bacterial infection triggers the transition from fresh thromboembolic material to fibrotic tissue in CTEPH.
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Methods |
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Tissues
We collected thromboemboli from 19 consecutive CTEPH patients undergoing classical PEA23 between July 2005 and December 2006 at our institution, and examined all (n=7) surgical specimens from VA-shunt/infected pacemaker carriers in the CTEPH tissue database since 1994. Because of the limited number of acute pulmonary thrombectomies at our institution, only 5 acute pulmonary thromboemboli were available for analysis (Figure 2B, lanes 1' to 5'). Therefore, we also collected fresh varicophlebitis thrombi from patients undergoing varicose vein surgeries (n=23).24 These samples were used as reference standards. Tissue aliquots were immediately fixed in 7.5% buffered formaldehyde and frozen in liquid nitrogen (LN2). Patients and controls gave written informed consent under a study that was approved by the Ethics Committee of the Medical University of Vienna.
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DNA Extraction and Staphylococcus-Specific Polymerase Chain Reaction
DNA was purified from chronic (n=26) and acute (n=28) thrombi using the QIAamp DNA Mini kit (Qiagen). Staphylococcus-specific polymerase chain reaction (PCR) was performed in the GeneAmp PCR System 2400. Bacterial 16S ribosomal DNA was amplified using the genus-specific forward primer 5'-CAGCTCGTGTCGTGAGATGT-3' and reverse primer 5'-CCAATCATTTGTCCCACCTTCG-3'. The thermal profile consisted of an initial hot start at 95°C for 5 min followed by 35 cycles of 40 s at 94°C, 60 s at 56°C, 60 s at 72°C, and a final elongation step at 72°C for 7 min. The resulting amplicon of about 420 bp contains hypervariable regions allowing for species-level identification. A negative control reaction with deionized water was included in each series of PCR reactions. S aureus DNA was used as a positive control. Sequences from cloned PCR products were compared with the GenBank database using the Advanced Gapped BLAST program. Special care was taken to avoid contamination of the samples with environmental bacterial DNA and amplicons. Pre-PCR facilities were kept strictly separated from the post-PCR areas. All PCR reactions were set up in a designated hood where UV light irradiation was used to destroy possible contaminants in the reagents and equipment. Multiple samples were taken from different areas of the specimens for DNA extraction.
Bacterial Cultures
A piece of CTEPH or control specimen was collected in sterile saline and inoculated into one aerobic pediatric blood culture bottle (BactAlert PF flask, 4 to 10 mg of material under sterile conditions). Another part of the sample was inoculated into the ISOLATOR-system (Oxoid), a highly sensitive lysis-centrifugation system, allowing not only detection of conventional, easy-to-grow microorganisms, but also of fastidious and intracellular pathogens.
RNA Preparation and Real Time PCR
After RNA isolation and cDNA synthesis, quantitative fluorogenic PCR was performed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Specific TaqMan primers and probes for human CTGF (TaqMan Gene Expression Assay, Applied Biosystems, Assay ID Hs00170014_m1), human TGF-β (Applied Biosystems, Assay ID Hs99999918_m1), and eukaryotic 18S rRNA endogenous control were used. PCR was performed under standard conditions: ie, 40 cycles of denaturation at 95°C for 15 s and annealing/elongation at 60°C for 1 min. The mRNA expression levels of the genes were normalized to endogenous 18S-RNA levels. In parallel, the same RNA samples were used as a template for RT-PCR amplifications using Staphylococcus-specific 16S rDNA primers.
Preparation of S aureus Suspension
S aureus ATCC 12600 (American Type Culture Collection, USA) was cultivated on Columbia blood agar plates (Becton Dickinson) for 24 h at 35°C before injection. The bacterial suspension was dissolved in sodium chloride. Using spectrophotometry and the McFarland index, an optical density at 620 nm corresponding to 1.5 to 5x108 cells per ml was achieved for the final suspension.
Murine Model of Inferior Vena Cava Thrombosis
Adult female Balb/c mice (n=64, age: 8 weeks, weight: 18 to 20g, Himberg, Vienna) were anesthetized with 100 mg/kg ketamine (Ketavet) and 5 mg/kg xylacine (Rompun) i.p. The IVC was exposed below the renal veins through a midline laparotomy incision. The intestines were retracted, and retroperitoneal blunt dissection of the infrarenal vena cava was performed to mobilize a 5-mm segment distal to the left renal vein. In contrast to the original description,25 endothelial denudation with a neurosurgical vascular clip was not performed because of a high rate of rupture of the IVC wall with this method. In our hands, injury to the endothelial layer that occurred as a consequence of the manipulation was a sufficient prothrombotic stimulus. A 5-0 Prolene suture was placed alongside the vena cava. A stenosis was produced in the vein by tying a 4-0 silk suture around the IVC to include the Prolene suture. The Prolene was then pulled to allow blood to continue to pass up the vein. The intestines were replaced, and the abdominal wall was sutured. The animals were allowed to recover from anesthesia. Pain was treated by 0.05 mg/kg buprenorphine (Temgesic) s.c., every 8 hours. Mice had access to water ad libitum and conventional commercial chow.
For induction of infected thrombi, mice (8 per time point) were injected via the tail vein on day 1 after IVC ligation with 20 mg/kg of S aureus (ATCC 12600, Staphylococcus aureus subsp. aureus Rosenbach) dissolved in 200 µL saline. Animals were euthanized with an overdose of pentobarbital (Vetanarcol) on days 3, 7, 14, and 28 after ligation. Control mice received an equivalent volume of saline alone. Thrombi were obtained in every single animal. IVCs including thrombi were carefully harvested. For sizing, the thrombus was considered to be a cylinder, and volumes were calculated by the formula: thrombus radius2 x 
x thrombus length. Thrombus radius and length were measured with a caliper. After weighing, thrombi were fixed in 7.5% buffered formaldehyde. Thrombi from each animal were cut into 2-mm tissue blocks and embedded in paraffin in a transverse orientation. Paraffin blocks were cut in 3-µm sections along the cranio-caudal extension. Cross-sectional areas were determined of all sections taken through the entire thrombus. For the figure, serial sections at random cross-sectional areas were stained. In a separate series of experiments, thrombi were immediately frozen in LN2 and stored at –180°C for molecular analyses. For thrombus area measurements an Olympus BX 50 microscope equipped with the imaging software Axio (Version 3.0 to 2002, Carl Zeiss Vision GmbH) was used. Two independent observers blinded to the experimental groups performed cross-sectional planimetry. Specific TaqMan primers and probes for murine TGF-β (Applied Biosystems, Assay ID Mm00441724_m1) and CTGF (Applied Biosystems, Assay ID Mm00515790_g1) were used. The Austrian Ministry of Science approved the animal studies.
Quantification of Collagen
Collagen quantification was performed as previously described.26 In brief, the methodology relies on the binding of SiriusRed (Sigma) to collagen, and FastGreen (Merck) to noncollagenous components. Both dyes are eluted, and the absorbances obtained at 540 and 605 nm can be used to estimate the amount of collagen and total protein. Collagen content is expressed as collagen in percent of total protein content.
(Immuno-) Histochemistry
For histochemical analyses, a modified Trichrome stain was used.27 Immunohistochemical analyses were performed as described.11 Purified antimouse neutrophil monoclonal antibody (CL8993AP, Cedarlane Laboratories), monoclonal antihuman 
-smooth muscle actin (-SMA) antibody (M851, DAKO), monoclonal antibody against peptidoglycan (2E9, from Laman JD, Department of Immunology, Erasmus MC, Rotterdam, The Netherlands), antihuman vimentin antibody (RM-9120-SO, NeoMarkers), and antimouse F4/80 glycoprotein antibody (14-4801, eBioscience) were used.
Statistics
The significance of intergroup differences was determined by ANOVA with repeated measures. All results are expressed as mean±SD. Nominal variables were compared with the Chi square test. The Spearman test was used for correlation of continuous variables. P<0.05 was considered statistically significant.
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Results |
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Patient Characteristics
Mean age of CTEPH patients was 50±17 years, donors of control thrombi were 55±12 years old (P=0.15). There were 9 males among the CTEPH patients, and 12 males among donors of fresh clots. Seven CTEPH patients carried either a VA-shunt or pacemaker, and 5 of those had a history of single or recurrent shunt/lead infection with positive blood/atrial thrombus cultures at the time of a clinically apparent infection (Table). Device implantation occurred 103±87 months before CTEPH diagnosis. Of the controls with acute pulmonary embolism, 1 patient had papillary bladder tumor, 1 suffered from perforated stomach ulcer, 1 female patient was an obese smoker, and 2 patients had recently undergone major surgery. Patients had no overt signs of infection at the time of CTEPH diagnosis. Mean C-reactive protein was 1.1±0.8 mg/dL, blood leukocyte count was 9.3±4.7G/L, and fibrinogen was 382±103 mg/dL, compared with a C-reactive protein of 3.6±4.6 mg/dL, (P=0.09), a blood leukocyte count of 11.6±6.5G/L, (P=0.08), and fibrinogen of 443±157 mg/dL (P=0.07) in controls.
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Characterization of CTEPH Thrombi
Both macroscopically (Figure 1A and 1B) and by histological analysis, acute pulmonary emboli differ from CTEPH thrombi. Reddish soft casts of the pulmonary arterial tree in acute pulmonary embolism contrast the fibrotic whitish material of CTEPH. Trichrome-stained paraffin sections (Figure 1C and 1D) illustrate fresh fibrin strands with scattered inflammatory cells as their main components, whereas CTEPH specimens are mainly composed of collagen and -SMA-positive cells.
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The relative quantity of CTGF mRNA as a molecular marker of fibrosis was elevated in CTEPH thromboemboli compared with acute pulmonary thrombi (0.23±0.32 versus 1.69±0.98 on a logarithmic scale, P=0.013, Figure 1E). The relative quantity of TGF-β mRNA was low in CTEPH specimens (0.84±0.61), compared with acute thrombi (2.06±0.41, P=0.006, Figure 1F).
Human CTEPH Specimens Contain Staphylococcus-Specific DNA
Whereas control samples (n=28) were negative, a Staphylococcus-specific 420-bp product was present in 7 of 26 CTEPH thrombi (or 6 of 7 VA-shunt/pacemaker carriers, P=0.019, Figure 2). All but 1 patient in whom Staphylococcus-specific DNA was detected were VA-shunt or pacemaker carriers with a history of device infection (Table). Sequencing revealed the presence of S aureus in 5 cases, and S epidermidis in 1 case (Table). A comparison between bacterial DNA positive and negative human CTEPH samples revealed a statistically significant difference in collagen content (10.52±5.11 versus 5.23±3.68% collagen, P=0.036; Figure 3A) and in the relative CTGF mRNA levels (1.61±0.59 versus 0.83±0.56, P=0.027; Figure 3B). Furthermore, a strong correlation was found between thrombus CTGF mRNA levels and thrombus collagen content (R2=0.853, P<0.001; Figure 3C).
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Delayed Resolution of Murine Thrombi After Infection With S aureus
To study thrombus resolution, we used a mouse model of IVC thrombosis.25,28 After infection, Staphylococcus-specific DNA was detectable in all thrombus specimens at all time points by culture and specific RT-PCR from infected mice, but in none of the control mice (data not shown). Thrombi from infected mice were significantly larger than thrombi from uninfected animals, with larger cross-sectional thrombus areas (Figure 4A through 4H, and Figure 5C, P<0.001), thrombus volumes (Figure 5A, P<0.001) and weights (Figure 5B, P=0.037). In addition, bacterial infection was associated with an increase in the relative area of collagen, ie, the ratio between the green collagen-containing area and the total thrombus cross-sectional area (Figure 4A through 4H, and Figure 5D, P<0.001). A marked increase in -SMA immunoreactive, vimentin-positive cells was noted on days 14 and 28 after infection (Figure 4I through 4P, and Figure 5E, P<0.001), as well as an increased neutrophil cell count per mm2 cross sectional thrombus area (Figure 5F, P<0.001). By contrast, macrophage counts were lower in infected thrombi than in control samples (P=0.001, data not shown), most prominently by day 14 after vena cava ligation. The relative CTGF mRNA level was elevated on day 28 after infection (Figure 5G, P<0.001). By comparison, relative TGF-β mRNA levels were elevated by day 7 after bacterial infection (Figure 5H, P<0.001).
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Discussion |
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The generation of thrombin from its precursor prothrombin is the central event of blood coagulation, which is essential to hemostasis and the culprit in thrombosis. This is a highly regulated, dynamic, and rapid process. By contrast, the timely removal of thrombus results from the concerted action of plasmatic fibrinolysis and a complex vascular remodeling process,29 which is time-consuming and involves circulating cells and cells of the vessel wall.30 Compared with the scientific interest in mechanisms of thrombus formation,31 few studies have addressed the vascular biology of thrombus resolution. We hypothesized that CTEPH is a disorder of misguided thrombus resolution.
CTEPH evolves over years, and decades may lie between the initiating thromboembolic event, the onset of symptoms and diagnosis4 (Table). Thus, CTEPH specimens represent the end-stage of a vascular remodeling process involving the intimal and medial layers of an affected pulmonary artery. In fact, CTEPH thrombi markedly differ from acute pulmonary emboli,5 and also from chronic pulmonary arterial thrombi seen for example in about 20% of Eisenmenger patients, as a consequence of chronic slow flow attributable to depressed right ventricular function.32 New vessel formation and fibrosis are histological hallmarks of CTEPH thrombi.11 Given the uncertainties regarding a simple mechanistic thromboembolic origin of CTEPH,10 a specific in situ vascular remodeling process is proposed.33
All but one Staphylococcus DNA-positive CTEPH case in this study were VA-shunt/pacemaker carriers, and all but one had had positive bacterial cultures previously, either from right atrial thrombi or from whole blood (Table). By contrast, none of 28 fresh venous thrombi were culture-positive for S aureus or epidermidis or contained staphylococcal DNA. Neither patients nor controls had clinical signs of active infection at the time of sampling, nor was there evidence for active bacterial metabolism based on bacterial cultures of CTEPH specimens, peptidoglycan immunostaining, and S aureus RNA analyses (data not shown). Mildly increased C-reactive protein levels in patients #1, 3, 5, and 6 (Table) without concomitant leukocytosis are more likely attributable to the inflammatory state associated with chronic heart failure34 than a sign for an active bacterial infection. These data suggest that bacterial DNA within chronic thrombi represents remnants of previous infection.
A tight correlation between the amount of bacterial DNA and the degree of fibrosis or CTGF mRNA seems unlikely, because pathogen virulence as well as host susceptibility factors determine the extent of infectious disease.35 However, the comparison between bacterial DNA positive and negative human CTEPH samples revealed a significant difference in collagen content and relative CTGF mRNA levels (Figure 3), strengthening the concept that bacterial infection is a major determinant of thrombus fibrosis and remodeling.
Human thrombi were characterized by features of a fibrotic process36 (Figure 1) with an increased collagen content compared with fresh thrombi. CTGF mRNA was found significantly elevated by day 28 (Figure 5G) in the model, which is considered the most relevant time point for this comparison, and a strong CTGF signal was detected in human CTEPH samples (Figure 1E). By contrast, TGF-β mRNA was markedly lower in CTEPH specimens than in acute thrombi (Figure 1F). These findings are in accord with TGF-β expression patterns in a variety of wound healing and fibrosis models.37 On injury, TGF-β is rapidly induced and may induce CTGF expression.38 Hypoxia-inducible factor 1 that is activated on bacterial infection39 induces CTGF expression, possibly via a hypoxia-responsive element in the CTGF promotor.40 Peptidoglycan from S aureus causes rapid release of tumor necrosis factor (TNF)- and other inflammatory cytokines resulting in TGF-β induction.41 Similarly, S aureus protein A, a highly expressed surface component, can mimic TNF- to activate its receptor and initiate subsequent proinflammatory42 and profibrotic signaling.43
Later in the course of healing, TGFβ expression is confined to the leading edge of the scar tissue.22 The differences in the TGF-β expression profile between human CTEPH thromboemboli and infected murine thrombi illustrate one of the limitations of the model. An observation period of 28 days is contrasted by a time interval of 103±87 months between device implantation and the diagnosis of CTEPH. In addition, vascular remodeling in pulmonary vessels may be subjected to different rules than in the caval vein. In particular, it has to be taken into account that bacterial infection in the mouse model involved the entire wall of the IVC, resulting in a thrombophlebitis.
A decreased neutrophil count in the early thrombotic process is associated with increased collagen and impaired thrombus resolution, which was not observed when occurring 24 h after thrombus formation.44 In our model, bacterial injection was performed 24 h after thrombus formation, confirming that the role of neutrophils in thrombus resolution is time-dependent.
Another major finding was that macrophages that appear to be essential for thrombus recanalization and resolution25,45 were significantly reduced in number in infected murine thrombi compared with controls. In vitro, monocytes/macrophages have been shown to undergo apoptosis within 2 to 4 h after phagocytosis of S aureus.46
Based on the analysis of patients with a history of intravenous catheter infection, we conclude that CTEPH is a disorder of misguided thrombus resolution with disproportionate fibrosis that may be driven by S aureus infection.
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Acknowledgments |
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Sources of Funding
This research project received financial support from the European Commission under the 6th Framework Programme (Contract No: LSHM-CT-2005-018725, PULMOTENSION). In addition, this research was in part supported by FWF S9406-B11 (to I.M.L.), and the Österreichischer Herzfonds (to D.B.).
Disclosures
None.
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Footnotes |
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Original received September 13, 2007; final version accepted January 20, 2008.
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References |
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1. Menendez R, Nauffal D, Cremades MJ. Prognostic factors in restoration of pulmonary flow after submassive pulmonary embolism: a multiple regression analysis. Eur Respir J. 1998; 11: 560–564.[Abstract]
2. Fedullo PF, Auger WR, Kerr KM, Rubin LJ. Chronic thromboembolic pulmonary hypertension. N Engl J Med. 2001; 345: 1465–1472.
3. Pengo V, Lensing AW, Prins MH, Marchiori A, Davidson BL, Tiozzo F, Albanese P, Biasiolo A, Pegoraro C, Iliceto S, Prandoni P. Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. N Engl J Med. 2004; 350: 2257–2264.
4. Moser KM, Auger WR, Fedullo PF. Chronic major-vessel thromboembolic pulmonary hypertension. Circulation. 1990; 81: 1735–1743.
5. Lang IM. Chronic thromboembolic pulmonary hypertension–not so rare after all. N Engl J Med. 2004; 350: 2236–2238.
6. Wolf M, Boyer-Neumann C, Parent F, Eschwege V, Jaillet H, Meyer D, Simonneau G. Thrombotic risk factors in pulmonary hypertension. Eur Respir J. 2000; 15: 395–399.[Abstract]
7. Olman MA, Marsh JJ, Lang IM, Moser KM, Binder BR, Schleef RR. Endogenous fibrinolytic system in chronic large-vessel thromboembolic pulmonary hypertension. Circulation. 1992; 86: 1241–1248.
8. Lang IM, Marsh JJ, Olman MA, Moser KM, Schleef RR. Parallel analysis of tissue-type plasminogen activator and type 1 plasminogen activator inhibitor in plasma and endothelial cells derived from patients with chronic pulmonary thromboemboli. Circulation. 1994; 90: 706–712.
9. Marsh JJ, Konopka RG, Lang IM, Wang HY, Pedersen C, Chiles P, Reilly CF, Moser KM. Suppression of thrombolysis in a canine model of pulmonary embolism. Circulation. 1994; 90: 3091–3097.
10. Egermayer P, Peacock AJ. Is pulmonary embolism a common cause of chronic pulmonary hypertension? Limitations of the embolic hypothesis. Eur Respir J. 2000; 15: 440–448.[Abstract]
11. Lang IM, Marsh JJ, Olman MA, Moser KM, Loskutoff DJ, Schleef RR. Expression of type 1 plasminogen activator inhibitor in chronic pulmonary thromboemboli. Circulation. 1994; 89: 2715–2721.
12. Bonderman D, Jakowitsch J, Adlbrecht C, Schemper M, Kyrle PA, Schonauer V, Exner M, Klepetko W, Kneussl MP, Maurer G, Lang I. Medical conditions increasing the risk of chronic thromboembolic pulmonary hypertension. Thromb Haemost. 2005; 93: 512–516.[Medline] [Order article via Infotrieve]
13. Bonderman D, Skoro-Sajer N, Jakowitsch J, Adlbrecht C, Dunkler D, Taghavi S, Klepetko W, Kneussl M, Lang IM. Predictors of outcome in chronic thromboembolic pulmonary hypertension. Circulation. 2007; 115: 2153–2158.
14. Schoenbaum SC, Gardner P, Shillito J. Infections of cerebrospinal fluid shunts: epidemiology, clinical manifestations, and therapy. J Infect Dis. 1975; 131: 543–552.[Medline] [Order article via Infotrieve]
15. Colli BO, Starr EM, Martelli N. [Surgical treatment of hydrocephalus in children. II. Complications]. Ar Qneuropsiquiatr. 1981; 39: 408–419.
16. Rockey DC. Hepatic fibrogenesis and hepatitis C. Semin Gastrointest Dis. 2000; 11: 69–83.[Medline] [Order article via Infotrieve]
17. Vassallo R. Viral-induced inflammation in interstitial lung diseases. Semin Respir Infect. 2003; 18: 55–60.[CrossRef][Medline] [Order article via Infotrieve]
18. Li Z, Li J. Local expressions of TGF-β1, TGF-β1RI, CTGF, and Smad-7 in Helicobacter pylori-associated gastritis. Scand J Gastroenterol. 2006; 41: 1007–1012.[CrossRef][Medline] [Order article via Infotrieve]
19. Meneghin A, Hogaboam CM. Infectious disease, the innate immune response, and fibrosis. J Clin Invest. 2007; 117: 530–538.[CrossRef][Medline] [Order article via Infotrieve]
20. Bannerman DD, Paape MJ, Chockalingam A. Staphylococcus aureus intramammary infection elicits increased production of transforming growth factor-, β1, and β2. Vet Immunol Immunopathol. 2006; 112: 309–315.[CrossRef][Medline]
[Order article via Infotrieve]
21. Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, Grotendorst GR. Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: down-regulation by cAMP. Faseb J. 1999; 13: 1774–1786.
22. Leask A, Abraham DJ. TGF-β signaling and the fibrotic response. FASEB J. 2004; 18: 816–827.
23. Jamieson SW. Pulmonary thromboendarterectomy. Heart. 1998; 79: 118–120.
24. Verrel F, Ruppert V, Spengel FA, Steckmeier B. Stage-adapted therapy concept in ascending thrombophlebitis. Zentralbl Chir. 2001; 126: 531–536.[CrossRef][Medline] [Order article via Infotrieve]
25. Singh I, Burnand KG, Collins M, Luttun A, Collen D, Boelhouwer B, Smith A. Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells. Circulation. 2003; 107: 869–875.
26. Lopez-De Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem. 1985; 33: 737–743.[Abstract]
27. Garvey W, Fathi A, Bigelow F, Carpenter B, Jimenez C. A combined elastic, fibrin and collagen stain. Stain Technol. 1987; 62: 365–368.[Medline] [Order article via Infotrieve]
28. Singh I, Smith A, Vanzieleghem B, Collen D, Burnand K, Saint-Remy JM, Jacquemin M. Antithrombotic effects of controlled inhibition of factor VIII with a partially inhibitory human monoclonal antibody in a murine vena cava thrombosis model. Blood. 2002; 99: 3235–3240.
29. Lang IM, Moser KM, Schleef RR. Elevated expression of urokinase-like plasminogen activator and plasminogen activator inhibitor type 1 during the vascular remodeling associated with pulmonary thromboembolism. Arterioscler Thromb Vasc Biol. 1998; 18: 808–815.
30. Modarai B, Burnand KG, Sawyer B, Smith A. Endothelial progenitor cells are recruited into resolving venous thrombi. Circulation. 2005; 111: 2645–2653.
31. Cushman M. Epidemiology and risk factors for venous thrombosis. Semin Hematol. 2007; 44: 62–69.[CrossRef][Medline] [Order article via Infotrieve]
32. Broberg CS, Ujita M, Prasad S, Li W, Rubens M, Bax BE, Davidson SJ, Bouzas B, Gibbs JS, Burman J, Gatzoulis MA. Pulmonary arterial thrombosis in eisenmenger syndrome is associated with biventricular dysfunction and decreased pulmonary flow velocity. J Am Coll Cardiol. 2007; 50: 634–642.
33. Hoeper MM, Mayer E, Simonneau G, Rubin LJ. Chronic thromboembolic pulmonary hypertension Circulation. 2006; 113: 2011–2020.
34. Petersen JW, Felker GM. Inflammatory biomarkers in heart failure. Congest Heart Fail. 2006; 12: 324–328.[CrossRef][Medline] [Order article via Infotrieve]
35. Brogden KA, Roth JA, Stanton TB, Bolin CA, Minion FC, Wannemuehler MJ. Virulence Mechanisms of Bacterial Pathogens. Washington, DC: ASM Press; 2000.
36. Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest. 2007; 117: 524–529.[CrossRef][Medline] [Order article via Infotrieve]
37. Khalil N, Xu YD, O’Connor R, Duronio V. Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor-β1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK. J Biol Chem. 2005; 280: 43000–43009.
38. Grotendorst GR, Okochi H, Hayashi N. A novel transforming growth factor β response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ. 1996; 7: 469–480.[Abstract]
39. Kempf VA, Lebiedziejewski M, Alitalo K, Walzlein JH, Ehehalt U, Fiebig J, Huber S, Schutt B, Sander CA, Muller S, Grassl G, Yazdi AS, Brehm B, Autenrieth IB. Activation of hypoxia-inducible factor-1 in bacillary angiomatosis: evidence for a role of hypoxia-inducible factor-1 in bacterial infections. Circulation. 2005; 111: 1054–1062.
40. Hong KH, Yoo SA, Kang SS, Choi JJ, Kim WU, Cho CS. Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts. Clin Exp Immunol. 2006; 146: 362–370.[CrossRef][Medline] [Order article via Infotrieve]
41. Aasen AO, Wang JE. Mediator responses in surgical infections. Surg Infect (Larchmt). 2006; 7 Suppl 2: S3–S4.[CrossRef]
42. Gomez MI, O’Seaghdha M, Magargee M, Foster TJ, Prince AS. Staphylococcus aureus protein A activates TNFR1 signaling through conserved IgG binding domains. J Biol Chem. 2006; 281: 20190–20196.
43. Guo G, Morrissey J, McCracken R, Tolley T, Klahr S. Role of TNFR1 and TNFR2 receptors in tubulointerstitial fibrosis of obstructive nephropathy. Am J Physiol. 1999; 277: F766–F772.[Medline] [Order article via Infotrieve]
44. Henke PK, Varma MR, Deatrick KB, Dewyer NA, Lynch EM, Moore AJ, Dubay DA, Sukheepod P, Pearce CG, Upchurch GR Jr, Kunkel SL, Franz MG, Wakefield TW. Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization. Thromb Haemost. 2006; 95: 272–281.[Medline] [Order article via Infotrieve]
45. Ali T, Humphries J, Burnand K, Sawyer B, Bursill C, Channon K, Greaves D, Rollins B, Charo IF, Smith A. Monocyte recruitment in venous thrombus resolution. J Vasc Surg. 2006; 43: 601–608.[CrossRef][Medline] [Order article via Infotrieve]
46. Baran J, Guzik K, Hryniewicz W, Ernst M, Flad HD, Pryjma J. Apoptosis of monocytes and prolonged survival of granulocytes as a result of phagocytosis of bacteria. Infect Immun. 1996; 64: 4242–4248.[Abstract]
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