Pulmonary embolism (PE) results from deep vein thrombosis (DVT) and can lead to chronic thromboembolic pulmonary hypertension (CTEPH) involving vascular dysfunction. Mechanisms are incompletely understood, in part due to lack of mouse models. We induced PE in C57BL/6 mice by intravenous injection of thrombin (166 U/kg BW), confirmed by a sudden bradycardia, bradypnea, and an increase in pulmonary artery (PA) pressure observed by high-frequency ultrasound. While symptoms resolved rapidly after single thrombin application, repeated PEs resulted in sustained PA-pressure increase, increased PA superoxide formation assessed by oxidative fluorescent microtopography, increased PA gp91phox expression, and endothelial dysfunction assessed by isometric tension studies of isolated PA segments after 24 hours. DVT was modeled in C57BL/6 mice by ligation of the inferior vena cava (IVC). Importantly, small pulmonary emboli could be detected along with a mild phenotype of PA endothelial dysfunction and oxidative stress in the absence of PA-pressure elevation. mRNA expression of plasminogen activator inhibitor-1 was increased in PAs of mice with recurrent PE after repetitive thrombin injections and to a lesser extent in DVT mice. In summary, our data suggest that PA endothelial dysfunction, induced by gp91phox-derived ROS, is an early event upon repetitive PE. This phenomenon might help to elucidate the mechanisms of PA dysfunction in the pathogenesis of CTEPH.
Pulmonary arterial hypertension (PAH), defined as a mean pulmonary arterial pressure (PAP) of 25 mmHg or more, is a hemodynamic and pathophysiological state that can be found in multiple clinical conditions. Chronic thromboembolic pulmonary hypertension (CTEPH) is a major cause of pulmonary hypertension when it is not secondary to heart disease or chronic lung disease. It is believed that early in the disease, thrombotic material, mostly originating from large and deep veins in the lower extremities (deep vein thrombosis (DVT)), embolises into the pulmonary vasculature causing acute pulmonary embolism (PE). Acute PE mechanically obstructs pulmonary arteries and reduces the total diameter of the pulmonary vascular bed. The amount of blood that has to pass through the pulmonary vasculature is tied to the oxygen demand of the body and is therefore fixed to a certain range. In order for the same amount of blood to pass the decreased pulmonary vascular bed, a higher perfusion pressure is required and the PAP rises. While this elevation in PAP accompanies almost every PE exceeding a hemodynamically relevant size, PAP elevation is usually transient, as most patients are able to resolve the emboli with only minimal residual abnormalities. However, in up to 9.1% of patients with acute PEs and in over 10% of patients with recurrent PEs, the emboli are not completely resolved and persist in the major pulmonary arteries [
We therefore aimed to investigate the effect of single or repetitive sublethal experimental pulmonary embolism on the pulmonary vasculature and pulmonary endothelial dysfunction, further comparing it to the inferior vena cava-stenosis model, a widely used model to induce and study deep vein thrombosis (DVT) in mice [
All animal experiments were in accordance with the Declaration of Helsinki and National Institutes of Health guidelines. All experiments were approved by the Ethics Committee of the University Hospital Mainz and by the Institutional Animal Care and Use Committee (IACUC, Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany). All mice were housed in a barrier facility (Translational Animal Research Center (TARC), University Medical Center Mainz), kept in filter top cages with 2–5 mice per cage under specific pathogen-free (SPF) conditions.
C56BL/6 mice originally obtained from Jackson Laboratories (C57BL/6J; Bar Harbor, USA) were used as experimental animals. After treatment, animals were anesthetized by isoflurane inhalation (5% inhalant in room air) and killed by exsanguination via right ventricular puncture. The heart and lungs were rapidly excised, transferred to 4°C Krebs-HEPES-solution (pH 7.35 containing NaCl 99.01 mM, KCl 4.69 mM, CaCl2 2.50 mM, MgSO4 1.20 mM, NaHCO3 25.0 mM, K2HPO4 1.03 mM, Na-HEPES 20.0 mM, and D-glucose 11.1 mM), and cleared of adhesive tissue.
To assess vasodilator properties of isolated pulmonary arterial segments, pulmonary arteries were cut into 3 mm segments and mounted on force transducers (Kent Scientific Corporation, Torrington, CT; PowerLab, ADInstruments, Spechbach, Germany) in organ chambers filled with Krebs–Henseleit solution (37°C, pH 7.35, containing 118 mM NaCl, 4.69 mM KCl, 1.87 mM CaCl2, 1.2 mM MgSO4, 1.03 mM K2HPO4, 25 mM NaHCO3, and 11.1 mM D-glucose) bubbled with carbogen gas (95% O2 and 5% CO2) and containing 1
Following preconstriction with phenylephrine (0.15
Male animals of at least 12 weeks of age with a minimum body weight of 25 g were anesthetized by intraperitoneal injection of a solution of midazolam (5 mg/kg; Ratiopharm GmbH, Ulm, Germany), medetomidine (0.5 mg/kg body; Pfizer Deutschland GmbH, Berlin, Germany), and fentanyl (0.05 mg/kg; Janssen-Cilag GmbH, Neuss, Germany). The animals were fixed on a custom-built stage and maintained at physiological temperature. For IVC flow restriction to induce DVT, animals were then depilated with hair removal cream at the area of surgery. A median laparotomy was performed, and the IVC was exposed by atraumatic surgery. We positioned a space holder (Asahi Fielder XT Guide Wire 0.014” [0.36 mm]; Abbot Vascular, Abbot Park, USA) on the outside of the vessel and placed a permanent narrowing ligature (7.0 monofil polypropylene filament, Prolene; Braun, Melsungen, Germany) exactly below the left renal vein. Subsequently, the wire was removed to avoid complete vessel occlusion. Side branches were left open in all groups [
Anesthesia of mice was induced in a chamber (2–4% isoflurane mixed with 0.2 L/min 100% O2) and maintained with a face mask (0.5–1.5% isoflurane with 0.05–0.1 L/min 100% O2). Animals were kept on a heated table mounted on a rail system (Visual Sonics, Toronto, Canada). Acute experimental pulmonary embolims were induced by retroorbital injection of 166 U/kg BW
Anesthesia of mice was induced in a chamber (2–4% isoflurane mixed with 0.2 L/min 100% O2) and maintained with a face mask (0.5–1.5% isoflurane with 0.05–0.1 L/min 100% O2). Animals were kept on a heated table mounted on a rail system (Visual Sonics, Toronto, Canada). Ultrasound was performed with the Vevo 770 System and a 40 MHz mouse scan head (RMV 706 for vascular sonography or RMV 707B for echocardiography; VisualSonics). Body temperature was monitored using a rectal probe and maintained at 37°C. The chest or the abdomen of the mouse was depilated, and warm ultrasound transmission gel was applied to enable visualization and optimize image quality. In the absence of a pulmonary valve stenosis or a right ventricular outflow tract obstruction, pulmonary arterial pressure (PAP) correlates to the right ventricular systolic pressure (RVSP). In mice, RVSP can be directly measured by invasive cannulation of the right ventricle via the jugular vein. This is a terminal procedure and therefore does not allow for the repetitive measurements required for our study. Estimation of PAP via the modified Bernoulli equation using the tricuspid valve regurgitation signal is not applicable, as mice do not exhibit a physiological tricuspid regurgitation like humans or larger animals. To overcome this, pulmonary arterial acceleration time (PAT) was assessed as a surrogate parameter for PAP/RVSP as described and verified by Thibault et al. [
IVC ligation results in IVC thrombus formation with small pulmonary embolisms, whereas intravenous thrombin injection results in large pulmonary embolisms with significantly reduced PA flow. HFUS Power-Doppler imaging (a) and pulsed-wave Doppler imaging (a) or histologic analysis (b) of the IVC revealed a thrombus formation upstream of the subtotal IVC ligation with no detectable flow (left), while no flow reduction or thrombus in the IVC was detectable after induction of experimental pulmonary embolisms after intravenous thrombin injection ((a), (b) right subpanel, (c) quantification). (d) Histologic analysis of pulmonary tissue revealed numerous large thromboemboli after thrombin injection ((d) right subpanels) which involved the large pulmonary arteries, while small thromboemboli involving the peripheral pulmonary arteries could be observed after IVC ligation ((d) left subpanels). After thrombin injection, a significant obstruction of the pulmonary arterial vascular bed ((e) right) along with a significant reduction of the PA flow ((f) right) could be observed. After IVC ligation, the degree of pulmonary arterial obstruction was significantly less ((e) middle) than in PE mice and did not result in an impaired PA flow ((f) middle). PA: pulmonary artery; 5–8 animals per group. Data are presented as mean and SEM. 1-way ANOVA with Bonferroni’s multiple comparison test.
For vascular sonography, first, a long-axis view was used to visualize the IVC, the ligation, and the formed thrombus. An optimal freeze-frame image was taken manually and, using the Vevo 770 software, the cross-sectional area of the clot was traced to obtain the measurement. The length, width, and area of clots were measured applying B-mode imaging.
For isolation of RNA, snap-frozen mouse pulmonary arteries were homogenized with stainless steel micropesteles (A. Hartenstein, Würzburg, Germany), and the modified guanidine isothiocyanate method of Chomczynski and Sacchi [
The lungs, hearts, and large vessels were fixed in situ by intratracheal instillation of paraformaldehyde (PFA) as described, embedded in paraffin, and sectioned. IVC thrombi were excised with the surrounding tissue and fixed, embedded, and serially sectioned at 5
Isolated pulmonary trunks and arteries were cut into rings and incubated in Krebs-HEPES solution for 15 min at 37°C, embedded in aluminium cups of about 1 mL of a polymeric resin (Tissue-Tek O.C.T. compound, Sakura Finetek, Staufen, Germany), and frozen in liquid nitrogen. Cryosections (6
Human pulmonary artery endothelial cells (HPAECs) were purchased from PromoCell (catalogue number C-12241; PromoCell, Heidelberg, Germany) and cultivated at 37°C under 5% CO2 in endothelial cell growth medium (PromoCell; catalogue number C-22010). ROS assays were performed as described [
For DCF enhanced fluorescence, the cells were switched to fresh medium containing 10
For DHE fluorescence, cells were switched to pre-warmed buffer containing 5
Data are expressed as mean ± SEM. Statistical calculations were performed with GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA). D’Agostino-and-Pearson normality test was first performed, and Pearson’s correlation, Fisher’s exact test, Mann–Whitney test, paired or unpaired
We performed IVC ligation to induce stenosis of the vein with 80% flow reduction and reproducibly induced DVT in male C57BL/6 mice [
Injection of thrombin not only caused PE but also led to hemodymic challenge of the right ventricle (RV), with dilatation of the RV, flattening of the intraventricular septum (so-called D-sign), and compression and compromise of the left ventricle (Figure
IVC ligation has no effect on pulmonary hemodynamics, while sublethal acute pulmonary embolism induced by intravenous thrombin injection results in pulmonary hypertension and right ventricular dysfunction. (a) left subpanel: B-mode imaging in parasternal short-axis view shows normal morphology of cardiac left and right ventricles (LV and RV, resp.), while noninvasive assessment of pulmonary arterial/right ventricular systolic pressure (RVSP) by measurement of pulmonary arterial acceleration time (PAT) did not reveal an increase in RVSP ((c) left subpanel). (b) left subpanel: representative PW-Doppler tracings 24 h after induction of IVC-thrombus formation by IVC ligation. Immediately after induction of sublethal acute PE by intravenous thrombin injection, cardiac imaging revealed RV enlargement ((a) right subpanel) correlating to a significantly decreased PAT indicating a significant increase in RVSP ((c) right subpanel, results are presented as PAT (ms) and as calculated RVSP
Next, we investigated the feasibility of our approach for induction of sustained pulmonary arterial hypertension (PAH). We repeatedly injected
Repeated thrombin injection led to recurrent PE and sustained PA pressure elevation and endothelial dysfunction. While after a single thrombin injection, symptoms were generally resolved and PA pressure normalized within minutes, the time to recovery lengthened with every injection. 24 h after (triple) pulmonary embolisms, PAT remained significantly shortened, indicating a persisting elevation of pulmonary pressure ((a) are presented as PAT (ms) and as calculated RVSP
It is well established that endothelial dysfunction can be caused by a dysequilibrium of increased superoxide formation and challenged scavenger systems, which results in loss of nitric oxide bioavailability. We therefore assessed vascular superoxide formation with dihydroxyethidium staining and expression of the most important vascular source of superoxide, the gp91phox NADPH oxidase. Repeated PEs induced a significant increase in pulmonary artery superoxide formation (Figures
Pulmonary arterial oxidative stress is mediated by gp91phox NADPH oxidase. (a and b) Microtopography revealed significantly increased superoxide levels in PAs from triple-embolised mice as compared to PAs from control mice or after IVC ligation. IHC (c) and qPCR (d) revealed a significantly increased NOX2/gp91phox expression in PAs after pulmonary embolism as compared to PAs from control mice or after IVC ligation. (e) qPCR of PAI-1 mRNA in PAs. Ctrl: control mice; AF: autofluorescence; DHE: dihydroethidium (a); gp91phox: antibody against gp91phox NADPH oxidase; DAPI: 4′,6-diamidin-2-phenylindol; IVCL: IVC ligated mice; APE: mice with recurrent acute PE. 5 animals per group; data are presented as mean and SEM. 1-way ANOVA and Bonferroni’s multiple comparison test.
Thrombin induces increased levels of reactive oxygen species (ROS) in pulmonary arterial endothelial cells (PAECs). Exposure of cultured human PAECs to thrombin resulted in increased levels of ROS as determined by the fluorescent probes DCF and DHE ((a, b) left representative images, (a, b) right quantification). Thr: thrombin. 9–12 high-power field acquisitions per group. Data are presented as mean and SEM. Mann–Whitney test.
The pathogenesis of CTEPH remains elusive; however, vascular dysfunction and remodelling as a sequel of repeated PE has been discussed as the main driver of the disease. It is well established that endothelial dysfunction (defined as a loss of nitric oxide bioavailability due to impaired biosynthesis and/or increased breakdown by superoxide radicals) is an early hallmark of systemic arterial disease in atherosclerosis, diabetes mellitus, or arterial hypertension [
In PAH, the potential role of endothelial dysfunction is less well understood. In hypoxia-induced pulmonary arterial hypertension, the remodelling of the pulmonary arteries has been ascribed to increased endothelial-to-mesenchymal transition of the pulmonary arterial endothelial cells in mice and humans [
DVT is the most common cause of pulmonary embolism, and ligation of the IVC to induce stenosis of the vein is currently regarded as the best mouse model to reflect human pathophysiology of DVT [
We have used a similar approach to induce PE, repeatedly injecting
We conclude that oxidative stress mediated by NADPH oxidase-produced superoxide anions and endothelial dysfunction of the pulmonary arteries are closely linked to PAH and occur early after pulmonary embolism. It even occurs in DVT in the absence of hemodynamically relevant PE, indicating a very early sign of vascular remodelling in pulmonary circulation in remote venous clotting. In summary, our study is the first to provide a link between pulmonary embolism, pulmonary arterial hypertension, and pulmonary arterial endothelial dysfunction and oxidative stress in mice. We established a new mouse model to induce PE that could help to develop a model of CTEPH in mice.
The authors declare that they have no conflicts of interest.
The authors are grateful for the expert technical assistance of Katharina Perius. This work contains results that Eleni Giokoglu performed in her undergraduate studies as an MD student for her thesis. This work was supported by grants of the German Federal Ministry of Education and Research (BMBF 01EO1003 and BMBF 01EO1503) to Moritz Brandt, Susanne H. Karbach, Katrin Schäfer, and Philip Wenzel and of the Boehringer Ingelheim Foundation to Susanne H. Karbach, Thomas Münzel, and Philip Wenzel and by the Center for Translational Vascular Biology of the University Medical Center Mainz.
Figure S1: Noninvasive assessment of PAP. To allow for repetitive measurements of PAP, pulmonary arterial acceleration time (PAT) was assessed as a surrogate parameter for PAP/RVSP as described and verified by Thibault et al. [