BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Localization of the NRAS:BCL-2 complex determines anti-apoptotic features associated with progressive disease in myelodysplastic syndromes.

We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.

Comparison of 18F-fluoro-deoxy-glucose, 18F-fluoro-methyl-choline, and 18F-DPA714 for positron-emission tomography imaging of leukocyte accumulation in the aortic wall of experimental abdominal aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a frequent form of atherothrombotic disease, whose natural history is to enlarge and rupture. Indicators other than AAA diameter would be useful for preventive surgery decision-making, including positron-emission tomography (PET) methods permitting visualization of aortic wall leukocyte activation relevant to prognostic AAA evaluation. In this study, we compare three PET tracers of activated leukocytes, 18F-fluoro-deoxy-glucose (FDG), 18F-fluoro-methyl-choline (FCH), and 18F-DPA714 (a peripheral benzodiazepine receptor antagonist) for in vivo PET quantification of aortic wall inflammation in rat experimental AAAs, in correlation with histopathological studies of lesions. METHODS: AAAs were induced by orthotopic implantation of decellularized guinea pig abdominal aorta in 46 Lewis rats. FDG-PET (n = 20), FCH-PET (n = 8), or both (n = 12) were performed 2 weeks to 4 months after the graft, 1 hour after tracer injection (30 MBq). Six rats (one of which had FDG-PET) underwent 18F-DPA714-PET. Rats were sacrificed after imaging; AAAs and normal thoracic aortas were cut into axial sections for quantitative autoradiography and histologic studies, including ED1 (macrophages) and CD8 T lymphocyte immunostaining. Ex vivo staining of AAAs and thoracic aortas with 18F-DPA714 and unlabeled competitors was performed. RESULTS: AAAs developed in 35 out of 46 cases. FCH uptake in AAAs was lower than that of FDG in all cases on imaging, with lower AAA-to-background maximal standardized uptake value (SUV(max)) ratios (1.78 ± 0.40 vs 2.71 ± 0.54; P < .01 for SUV(max) ratios), and lower AAA-to-normal aorta activity ratios on autoradiography (3.52 ± 1.26 vs 8.55 ± 4.23; P < .005). FDG AAA-to-background SUV(max) ratios correlated with the intensity of CD8 + ED1 staining (r = .76; P < .03). FCH AAA-to-background SUV(max) ratios correlated with the intensity of ED1 staining (r = .80; P < .03). 18F-DPA714 uptake was similar in AAAs and in normal aortas, both in vivo and ex vivo. CONCLUSIONS: In rat experimental AAA, characterized by an important aortic wall leukocytes activity, FDG-PET showed higher sensitivity than FCH-PET and 18F-DPA714-PET to detect activated leukocytes. This enhances potential interest of this tracer for prognostic evaluation of AAA in patients.

Non-invasive molecular imaging of fibrosis using a collagen-targeted peptidomimetic of the platelet collagen receptor glycoprotein VI.

BACKGROUND: Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III. METHODOLOGY/PRINCIPAL FINDINGS: An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10(-7) M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis. CONCLUSION/SIGNIFICANCE: Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.

Technetium 99m-labeled annexin V scintigraphy of platelet activation in vegetations of experimental endocarditis.

BACKGROUND: The pathophysiology of infective endocarditis involves a pathogen/host tissue interaction, leading to formation of infected thrombotic vegetations. Annexin V is a ligand of phosphatidylserines exposed by activated platelets and apoptotic cells. Because vegetations are platelet-fibrin clots in which platelet proaggregant activity is enhanced by bacterial colonization, we investigated the ability of annexin V labeled with technetium Tc 99m (99mTc-ANX) to provide functional imaging of these vegetations in experimental models of infective endocarditis. This ability was assessed in rabbits and rats because of the different interest of these 2 species in preclinical analysis. METHODS AND RESULTS: Nonbacterial thrombotic endocarditis was induced with the use of a catheter left indwelling through the aortic or tricuspid valve, and animals were injected with either a bacterial inoculum or saline. Scintigraphic investigations were performed 5 days later and showed a higher 99mTc-ANX uptake by vegetations in infected versus noninfected animals (ratio, 1.3 for in vivo acquisitions and 2 for autoradiography; P<0.0001 for all), whereas no significant uptake was present in controls. Right-sided endocarditis was associated with pulmonary uptake foci corresponding to emboli. Histological analysis of vegetations showed a specific uptake of 99mTc-ANX at the interface between circulating blood and vegetation. In parallel, underlying myocardial tissue showed myocyte apoptosis and mucoid degeneration, without extracellular matrix degradation at this stage. CONCLUSIONS: 99mTc-ANX is suitable for functional imaging of platelet-fibrin vegetations in endocarditis, as well as embolic events. 99mTc-ANX uptake reflects mainly platelet activation in the luminal layer of vegetations. This uptake is enhanced by bacterial colonization.

Myocardial uptake of 99mTc-annexin-V and 111In-antimyosin-antibodies after ischemia-reperfusion in rats.

OBJECTIVES: Phosphatidylserin exposure on cell surfaces occurs early during apoptosis and is detected in vivo by using (99m)Tc-annexin-V (ANX). Cardiomyocyte membrane disruption is detected in vivo by using (111)In-antimyosin-antibodies (AM). We aimed to determine if ANX and AM allow evaluation of the time-course of these two distinct cell death events after myocardial ischemia-reperfusion. METHODS: Coronary tying (20 min) followed by reperfusion (IR) was performed in 31 rats. Twelve of the rats were injected with ANX, 11 with AM, and eight with both tracers. Myocardial uptake of tracers was studied 1-2 h, 4 h, or 24 h after IR by scintigraphy (ANX, n = 14) and autoradiography (all cases), and compared to histology and Apostain staining. RESULTS: Scintigraphy was positive in all rats 2 h after IR and in three of five rats at 24 h. On autoradiography, ANX activity was intense in myocardial lesions as early as 1 h post-IR, whereas AM activity was mild at 2 h then increased at 4 h post-IR. ANX and AM uptakes evolved from mid-myocardium to endocardial and epicardial regions from 2 h to 24 h post-IR. Apostain staining was significant in myocardial lesions (p < 10(6) compared to six sham-operated rats). On histology, myocardial lesion was characterized by interstitial oedema, myocytes necrosis, and dramatic thinning at 24 h. CONCLUSION: These data suggest that ANX and AM allow temporal and regional evaluations of PS exposure and membrane disruption, respectively, during myocytes death after 20-min myocardial ischemia followed by reperfusion. Also, (i) apoptosis starts very early in injured myocardium, (ii) myocyte necrosis occurs later (3-4 h post-reperfusion), and (iii) most dead cells are removed from mid-myocardium between 6 h and 24 h after reperfusion.

Evaluation of 99mTc-UBI 29-41 scintigraphy for specific detection of experimental Staphylococcus aureus prosthetic joint infections.

PURPOSE: (99m)Tc-UBI 29-41 (UBI), an antimicrobial peptide, specifically targets bacteria. We tested the ability of UBI to discriminate between infected and uninfected prosthetic joints using a rabbit model previously validated. METHODS: Left knee arthroplasty was performed on 20 New Zealand rabbits, then 10(7) cfu of S. aureus (n = 12) or sterile saline (n = 8) was injected into the joint. On days 9 and 20 after surgery, planar UBI scintigraphy was performed in six infected and four uninfected rabbits, 1 h and 4 h p.i. (150 MBq), on a gamma camera. Operated-to-normal knee activity ratio (ONKR) was calculated on each scintigram. Then, after sacrifice, tissue samples of both knees were counted in a gamma counter. RESULTS: One rabbit injected with sterile saline had cutaneous infection at sacrifice and was excluded from analysis. ONKR was higher in infected than in uninfected animals 4 h p.i. 20 days after surgery: 1.75 +/- 0.48 vs 1.13 +/- 0.11, p = 0.04. From 1 h to 4 h p.i., ONKR increased in 9/12 infected and 0/7 uninfected animals. According to UBI uptake intensity and kinetics, scintigraphy was truly positive in all infected cases on day 9 and in four of six infected cases on day 20. It was truly negative in two of three sterile inflamed prosthetic knees on day 9, and in all cases on day 20. Biodistribution studies revealed increased UBI uptake in periprosthetic tissues in all animals 9 days after surgery, and only in infected animals on day 20. CONCLUSION: In this experimental study, (99m)Tc-UBI 29-41 scintigraphy permitted the early detection of acute prosthetic joint infection, and exclusion of infection in chronic sterile prosthetic joint inflammation.

99mTc-annexin-V functional imaging of luminal thrombus activity in abdominal aortic aneurysms.

BACKGROUND: The mural thrombus of abdominal aortic aneurysms (AAA) is involved in aneurysm progression via several interdependent biological processes including platelet activation. 99mTc-annexin V (ANX) is a scintigraphic tracer that binds to phosphatidylserine exposed on activated platelets and apoptotic cells. Here, we evaluated the potential of ANX imaging to assess mural thrombus biological activity in an experimental AAA model. The clinical applicability was further tested ex vivo on human samples of excised AAA thrombi. METHODS AND RESULTS: Experimental AAA was created by infusing elastase into infrarenal abdominal aorta in 17 rats, and 6 sham-operated rats were used as controls. Abdominal ANX scintigraphy was performed 2 weeks later followed by quantitative autoradiography and histological studies. Among the 13 rats which developed AAA, 11 displayed intense ANX uptake within AAA by scintigraphy. ANX uptake in the aneurysms on planar and single-photon emission computed tomography (SPECT) imaging was higher than that observed in infrarenal aorta of sham-operated controls (target/background ratio: 5.7+/-0.9 versus 1.33+/-0.21; P<0.005 for SPECT). Aneurysm-to-background activity ratios obtained by scintigraphy correlated with ANX activity in corresponding autoradiograms (R=0.69; P<0.02). This activity was located in the thrombus area where activated platelets and polymorphonuclear leukocytes accumulated. Similar patterns were also found in all of the 7 human AAA thrombi harvested during surgery. CONCLUSIONS: ANX imaging may assess mural thrombus renewal activity linked to permanent flowing blood interface.

(99m)Tc-annexin V and (111)In-antimyosin antibody uptake in experimental myocardial infarction in rats.

PURPOSE: (99m)Tc-annexin V (ANX) allows scintigraphic detection of apoptotic cells via specific binding to exposed phosphatidylserine. In myocardial infarction, apoptosis of myocytes is variable and depends especially on the presence or absence of coronary reperfusion. In this study, ANX uptake in non-reperfused experimental myocardial infarcts was compared with uptake of a marker of myocyte necrosis ((111)In-antimyosin antibodies, AM) and an immunohistochemical marker of apoptosis (Apostain). METHODS: The left anterior coronary artery was ligated in 47 Wistar rats, which were then injected with ANX (n=20), AM (n=21) or both (n=6). Myocardial uptake of ANX and AM was determined at 2 h (n=14), 4 h (n=14) and 24 h (n=19) after coronary ligation (CL), by quantitative autoradiography with (n=23) or without (n=24) gamma imaging. Heart-to-lung ratios (HLRs) and infarct-to-remote myocardium activity ratios (INRs) were calculated on the scintigrams and autoradiograms respectively. Cardiac sections were stained with haematoxylin-eosin and Apostain. The above studies were repeated in 12 normal rats. RESULTS: All rats with CL showed increased ANX and AM uptake in cardiac areas on scintigrams 24 h after CL, with HLRs higher than in controls: 3.1+/-0.6 versus 1.5+/-0.3 (p=0.001) for ANX and 1.99+/-0.44 versus 1.01+/-0.05 (p<0.0005) for AM. Autoradiography showed intense ANX and AM uptake in infarcts, with comparable topography and INRs at 2 h, 4 h and 24 h after CL (4.6+/-0.9 versus 5.0+/-1.8 at 24 h), while Apostain staining was very low (0.06+/-0.06% of cells). CONCLUSION: In this model of persistent CL, we observed increased ANX uptake in injured myocardium, comparable in intensity, topography and kinetics to that of AM. There was only minimal Apostain staining in the same areas.