Irene Neudorfer

Magnetic Resonance Imaging Contrast Agent Targeted Toward Activated Platelets Allows In Vivo Detection of Thrombosis and Monitoring of Thrombolysis

Background— Platelets are the key to thrombus formation and play a role in the development of atherosclerosis. Noninvasive imaging of activated platelets would be of great clinical interest. Here, we evaluate the ability of a magnetic resonance imaging (MRI) contrast agent consisting of microparticles of iron oxide (MPIOs) and a single-chain antibody targeting ligand-induced binding sites (LIBS) on activated glycoprotein IIb/IIIa to image carotid artery thrombi and atherosclerotic plaques. Methods and Results— Anti-LIBS antibody or control antibody was conjugated to 1-&mgr;m MPIOs (LIBS MPIO/control MPIO). Nonocclusive mural thrombi were induced in mice with 6% ferric chloride. MRI (at 9.4 T) was performed once before and repeatedly in 12-minute-long sequences after LIBS MPIO/control MPIO injection. After 36 minutes, a significant signal void, corresponding to MPIO accumulation, was observed with LIBS MPIOs but not control MPIOs (P<0.05). After thrombolysis, in LIBS MPIO–injected mice, the signal void subsided, indicating successful thrombolysis. On histology, the MPIO content of the thrombus, as well as thrombus size, correlated significantly with LIBS MPIO–induced signal void (both P<0.01). After ex vivo incubation of symptomatic human carotid plaques, MRI and histology confirmed binding to areas of platelet adhesion/aggregation for LIBS MPIOs but not for control MPIOs. Conclusions— LIBS MPIOs allow in vivo MRI of activated platelets with excellent contrast properties and monitoring of thrombolytic therapy. Furthermore, activated platelets were detected on the surface of symptomatic human carotid plaques by ex vivo MRI. This approach represents a novel noninvasive technique allowing the detection and quantification of platelet-containing thrombi.

Urine Proteome Analysis Reflects Atherosclerotic Disease in an ApoE−/− Mouse Model and Allows the Discovery of New Candidate Biomarkers in Mouse and Human Atherosclerosis

Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE−/− mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE−/− mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE−/− mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α1-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α1-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE−/− mice on HFD. Urinary excretion levels of collagen and α1-antitrypsin fragments also significantly correlated with intraplaque collagen and α1-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α1-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.

Dual-Contrast Molecular Imaging Allows Noninvasive Characterization of Myocardial Ischemia/Reperfusion Injury After Coronary Vessel Occlusion in Mice by Magnetic Resonance Imaging

Background— Inflammation and myocardial necrosis play important roles in ischemia/reperfusion injury after coronary artery occlusion and recanalization. The detection of inflammatory activity and the extent of myocardial necrosis itself are of great clinical and prognostic interest. We developed a dual, noninvasive imaging approach using molecular magnetic resonance imaging in an in vivo mouse model of myocardial ischemia and reperfusion. Methods and Results— Ischemia/reperfusion injury was induced in 10-week-old C57BL/6N mice by temporary ligation of the left anterior descending coronary artery. Activated platelets were targeted with a contrast agent consisting of microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody directed against a ligand-induced binding site (LIBS) on activated glycoprotein IIb/IIIa (LIBS-MPIOs). After injection and imaging of LIBS-MPIOs, late gadolinium enhancement was used to depict myocardial necrosis; these imaging experiments were also performed in P2Y12−/− mice. All imaging results were correlated to immunohistochemistry findings. Activated platelets were detectable by magnetic resonance imaging via a significant signal effect caused by LIBS-MPIOs in the area of left anterior descending coronary artery occlusion 2 hours after reperfusion. In parallel, late gadolinium enhancement identified the extent of myocardial necrosis. Immunohistochemistry confirmed that LIBS-MPIOs bound significantly to microthrombi in reperfused myocardium. Only background binding was found in P2Y12−/− mice. Conclusions— Dual molecular imaging of myocardial ischemia/reperfusion injury allows characterization of platelet-driven inflammation by LIBS-MPIOs and myocardial necrosis by late gadolinium enhancement. This noninvasive imaging strategy is of clinical interest for both diagnostic and prognostic purposes and highlights the potential of molecular magnetic resonance imaging for characterizing ischemia/reperfusion injury.

Molecular magnetic resonance imaging allows the detection of activated platelets in a new mouse model of coronary artery thrombosis.

Objective:The final event leading to myocardial infarction is adhesion and activation of platelets after rupture of an atherosclerotic plaque, ending in thrombotic occlusion of the coronary artery. Imaging of imminent vessel occlusion may improve patient care. The feasibility of molecular magnetic resonance imaging (MRI) for the detection of coronary artery thrombosis in mice was examined. Materials and Methods:The left anterior descending coronary artery was exposed by lateral thoracotomy and incubated with ferric chloride to induce nonocclusive thrombosis in C57Bl/6 mice. A single chain antibody targeting ligand-induced binding sites (LIBS) of the activated glycoprotein IIb/IIIa or control antibody was conjugated to 1 &mgr;m-sized microparticles of iron oxide (MPIOs), resulting in LIBS-MPIO or control-MPIO MRI contrast agent, and injected intravenously. Hearts were subjected to histology and ex vivo MRI at 9.4 Tesla. Results:Thrombus size was comparable in mice injected with control-MPIO and LIBS-MPIO in histology. Significant binding of MPIOs to thrombi was observed in LIBS-MPIO-injected animals while no binding was observed in control animals (P < 0.05). In MRI, LIBS-MPIO binding to thrombi of the left anterior descending coronary artery resulted in significant MPIO-induced signal void compared with controls (P < 0.05). MRI signal void and the amount of bound contrast agent particles in histology showed a significant positive linear correlation (r = 0.939, P < 0.001). Conclusions:We established a new mouse model of nonocclusive coronary artery thrombosis. LIBS-MPIO contrast agent binds to activated platelets in this model, allowing molecular MRI of coronary thrombosis. This could have important implications on the timely noninvasive detection of arterial thrombosis, helping to initiate early therapeutic interventions.

Dual Contrast Molecular Imaging Allows Noninvasive Characterization of Myocardial Ischemia/Reperfusion Injury After Coronary Vessel Occlusion in Mice by MRI

Background— Inflammation and myocardial necrosis play important roles in ischemia/reperfusion injury after coronary artery occlusion and recanalization. The detection of inflammatory activity and the extent of myocardial necrosis itself are of great clinical and prognostic interest. We developed a dual, noninvasive imaging approach using molecular magnetic resonance imaging in an in vivo mouse model of myocardial ischemia and reperfusion. Methods and Results— Ischemia/reperfusion injury was induced in 10-week-old C57BL/6N mice by temporary ligation of the left anterior descending coronary artery. Activated platelets were targeted with a contrast agent consisting of microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody directed against a ligand-induced binding site (LIBS) on activated glycoprotein IIb/IIIa (LIBS-MPIOs). After injection and imaging of LIBS-MPIOs, late gadolinium enhancement was used to depict myocardial necrosis; these imaging experiments were also performed in P2Y12−/− mice. All imaging results were correlated to immunohistochemistry findings. Activated platelets were detectable by magnetic resonance imaging via a significant signal effect caused by LIBS-MPIOs in the area of left anterior descending coronary artery occlusion 2 hours after reperfusion. In parallel, late gadolinium enhancement identified the extent of myocardial necrosis. Immunohistochemistry confirmed that LIBS-MPIOs bound significantly to microthrombi in reperfused myocardium. Only background binding was found in P2Y12−/− mice. Conclusions— Dual molecular imaging of myocardial ischemia/reperfusion injury allows characterization of platelet-driven inflammation by LIBS-MPIOs and myocardial necrosis by late gadolinium enhancement. This noninvasive imaging strategy is of clinical interest for both diagnostic and prognostic purposes and highlights the potential of molecular magnetic resonance imaging for characterizing ischemia/reperfusion injury.

Selenium supplementation induces metalloproteinase-dependent L-selectin shedding from monocytes

Selenium therapy in patients with severe sepsis improves clinical outcome and has been associated with increased activity of the selenoprotein glutathione peroxidase. However, the mechanism of the observed beneficial effects remains unclear. We determined the effect of selenium treatment on the monocyte adhesion molecule L‐selectin and L‐selectin‐related monocyte functions in vitro and transferred our findings to an in vivo mouse model. Monocytes were purified, cultured, and incubated in the presence or absence of supplemented selenium and metalloproteinase (MP) inhibitors for up to 16 h. Expression of L‐selectin was unaffected after 2 and 6 h but decreased after 16 h of incubation in the presence of selenium. Soluble L‐selectin (sL‐selectin) in the supernatant was determined by ELISA. A 2.3‐fold increase as a result of shedding of L‐selectin was observed after 16 h of selenium treatment. Addition of the MP inhibitors GM6001, TNF‐α‐converting enzyme inhibitor 2, or GW280264X strongly reduced selenium‐induced L‐selectin shedding, indicating a MP‐dependent mechanism. The functional consequences of L‐selectin shedding were examined in a flow chamber model. Selenium‐treated monocytes showed significantly decreased rolling and adhesion to the L‐selectin ligand Sialyl‐Lewisa under conditions of venous shear stress (0.5 dyne/cm2). Selenium treatment of C57BL6 mice led to increased serum levels of sL‐selectin, underscoring the in vivo relevance of our findings. We describe a selenium‐induced down‐regulation of L‐selectin on monocytes as a consequence of MP‐dependent shedding of this membrane‐anchored adhesion molecule. The impairment of monocyte adhesion by selenium supplementation may represent an important, underlying mechanism for the modulation of inflammatory reactions in patients with severe sepsis.

Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

Background Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. Methodology/Principal Findings LIBS as well as an unspecific control single-chain antibody were labeled with 111Indium (111In) via bifunctional DTPA ( = 111In-LIBS/111In-control). Autoradiography after incubation with 111In-LIBS on activated platelets in vitro (mean 3866±28 DLU/mm2, 4010±630 DLU/mm2 and 4520±293 DLU/mm2) produced a significantly higher ligand uptake compared to 111In-control (2101±76 DLU/mm2, 1181±96 DLU/mm2 and 1866±246 DLU/mm2) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of 111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630±10650 DLU/mm2 vs. 17390±7470 DLU/mm2; P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with 111In-LIBS resulted in a significant increase of the target-to-background ratio compared to 111In-control (1.99±0.36 vs. 1.1±0.24; P<0.01). Conclusions/Significance Nuclear imaging with 111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application.

In vivo detection of activated platelets allows characterizing rupture of atherosclerotic plaques with molecular magnetic resonance imaging in mice

Background Early and non-invasive detection of platelets on micro atherothrombosis provides a means to identify unstable plaque and thereby allowing prophylactic treatment towards prevention of stroke or myocardial infarction. Molecular magnetic resonance imaging (mMRI) of activated platelets as early markers of plaque rupture using targeted contrast agents is a promising strategy. In this study, we aim to specifically image activated platelets in murine atherothrombosis by in vivo mMRI, using a dedicated animal model of plaque rupture. Methods An antibody targeting ligand-induced binding sites (LIBS) on the glycoprotein IIb/IIIa-receptor of activated platelets was conjugated to microparticles of iron oxide (MPIO) to form the LIBS-MPIO contrast agent causing a signal-extinction in T2*-weighted MRI. ApoE−/− mice (60 weeks-old) were fed a high fat diet for 5 weeks. Using a small needle, the surface of their carotid plaques was scratched under blood flow to induce atherothrombosis. In vivo 9.4 Tesla MRI was performed before and repetitively after intravenous injection of either LIBS-MPIO versus non-targeted-MPIO. Results LIBS-MPIO injected animals showed a significant signal extinction (p<0.05) in MRI, corresponding to the site of plaque rupture and atherothrombosis in histology. The signal attenuation was effective for atherothrombosis occupying ≥2% of the vascular lumen. Histology further confirmed significant binding of LIBS-MPIO compared to control-MPIO on the thrombus developing on the surface of ruptured plaques (p<0.01). Conclusion in vivo mMRI detected activated platelets on mechanically ruptured atherosclerotic plaques in ApoE−/− mice with a high sensititvity. This imaging technology represents a unique opportunity for noninvasive detection of atherothrombosis and the identification of unstable atherosclerotic plaques with the ultimate promise to prevent strokes and myocardial infarctions.

Imaging monocytes with iron oxide nanoparticles targeted towards the monocyte integrin MAC‐1 (CD11b/CD18) does not result in improved atherosclerotic plaque detection by in vivo MRI

Imaging of macrophages with superparamagnetic iron oxide particles (SPIO) has been performed to improve detection of atherosclerotic plaque inflammation in human and mouse studies by molecular magnetic resonance imaging (MRI). Since affinity of the monocyte/macrophage integrin MAC-1 (CD11b/CD18) is upregulated in inflammation, we generated a contrast agent targeting CD11b (CD11b-SPIOs) for improved macrophage detection in plaques. CD11b-SPIOs and non-targeted SPIOs (control-SPIOs) were incubated in vitro with human monocytes/macrophages. As quantified by SPIO-induced MRI signal extinction, intracellular iron-content was significantly higher in monoytes/macrophages incubated with CD11b-SPIO than with control-SPIO in vitro (p < 0.05), suggesting an improved uptake of CD11b-SPIOs into monocytes. Therefore, the aortic arch (AA) and vessel branches of ApoE(-/-)-knockout mice on a Western-type diet were imaged before and 48 h after contrast agent injection of either CD11b-SPIOs or control-SPIOs, using a 9.4 T animal MRI system. The SPIO-induced change in the MRI signal was quantified, as well as the macrophage-content by anti-CD68 immunhistochemistry and the iron-content by Prussian-blue staining. However, SPIO-induced signal extinction in in vivo-MRI was similar in CD11b-SPIO and control-SPIO-injected animals, with a non-significant trend towards an improved uptake of CD11b-SPIOs in the subclavian artery and subsections of the AA. These data correlated well with the results obtained by histology. Although in vitro MRI-data indicated an increased uptake of targeted CD11b-SPIOs in monocytes/macrophages, in vivo mouse data do not allow improved atherosclerotic plaque detection compared WITH non-targeted SPIOs. Therefore, CD11b-targeted MRI contrast labelling of monocytes/macrophages does not seem to be a successful strategy in stable atherosclerotic plaques such as found in the ApoE(-/-)-knockout-model. However, the impressive correlation between MRI and histology data encourages further development of inflammation- and plaque-specific contrast agents for vulnerable plaque imaging.

Molecular Imaging of Activated Platelets Allows the Detection of Pulmonary Embolism with Magnetic Resonance Imaging.

Early and reliable detection of pulmonary embolism (PE) is critical for improving patient morbidity and mortality. The desire for low-threshold screening for pulmonary embolism is contradicted by unfavorable radiation of currently used computed tomography or nuclear techniques, while standard magnetic resonance imaging still struggles to provide sufficient diagnostic sensitivity in the lung. In this study we evaluate a molecular-targeted contrast agent against activated platelets for non-invasive detection of murine pulmonary thromboembolism using magnetic resonance imaging. By intravenous injection of human thrombin, pulmonary thromboembolism were consistently induced as confirmed by immunohistochemistry of the lung. Magnetic resonance imaging after thrombin injection showed local tissue edema in weighted images which co-localized with the histological presence of pulmonary thromboembolism. Furthermore, injection of a functionalized contrast agent targeting activated platelets provided sensitive evidence of focal accumulation of activated platelets within the edematous area, which, ex vivo, correlated well with the size of the pulmonary embolism. In summary, we here show delivery and specific binding of a functionalized molecular contrast agent against activated platelets for targeting pulmonary thromboembolism. Going forward, molecular imaging may provide new opportunities to increase sensitivity of magnetic resonance imaging for detection of pulmonary embolism.