Rationale: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE −/− ) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) ( P <0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE −/− mice were successfully identified by single-photon emission computed tomography imaging. 99m Tc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions: The 99m Tc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.

Human mesenchymal stem cells (hMSC) are a promising source for cell therapy after stroke. To deliver these cells, an IV injection appears safer than a local graft. We aimed to assess the whole-body biodistribution of IV-injected (99m)Tc-HMPAO-labeled hMSC in normal rats (n = 9) and following a right middle cerebral artery occlusion (MCAo, n = 9). Whole-body nuclear imaging, isolated organ counting (at 2 and 20 h after injection) and histology were performed. A higher activity was observed in the right damaged hemisphere of the MCAo group [6.5 +/- 0.9 x 10(-3) % of injected dose (ID)/g] than in the control group (3.6 +/- 1.2 x 10(-3) %ID/g), 20 h after injection. In MCAo rats, right hemisphere activity was higher than that observed in the contralateral hemisphere at 2 h after injection (11.6 +/- 2.8 vs. 9.8 +/- 1.7 x 10(-3) %ID/g). Following an initial hMSC lung accumulation, there was a decrease in pulmonary activity from 2 to 20 h after injection in both groups. The spleen was the only organ in which activity increased between 2 and 20 h. The presence of hMSC was documented in the spleen, liver, lung, and brain following histology. IV-injected hMSC are transiently trapped in the lungs, can be sequestered in the spleen, and are predominantly eliminated by kidneys. After 20 h, more hMSC are found in the ischemic lesion than into the undamaged cerebral tissue. IV delivery of hMSC could be the initial route for a clinical trial of tolerance.

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Adenosine and adenosine A(2A) receptor agonists have been shown to limit myocardial infarct size when given at vasodilatory doses during reperfusion. This beneficial effect is thought to be due, in part, to stimulation of adenosine A(2A) receptors on inflammatory cells. The specific aims of this study were to determine whether the anti-inflammatory and cardioprotective properties of a novel adenosine A(2A) receptor agonist, ATL-146e (ATL), alone or in combination with the phosphodiesterase IV inhibitor rolipram would occur using very low, nonvasodilating doses. In a canine model of reperfused myocardial infarction, low-dose ATL given alone reduced infarct size by 45% (P < 0.05 vs. control). When ATL was combined with a very low dose of rolipram (0.001 microg.kg(-1).min(-1)), a marked reduction in P-selectin expression and neutrophil infiltration (51% lower; P < 0.001 vs. control) was seen and the infarct size reduction (58% lower; P < 0.01 vs. control) was greater than observed with ATL (45% lower; P < 0.05) or rolipram (33% lower; P < 0.05) alone. In conclusion, a low, nonvasodilating dose of ATL, a highly selective adenosine A(2A) receptor agonist, reduced infarct size after reperfusion. Furthermore, combining ATL and the phosphodiesterase IV inhibitor rolipram reduced infarct size even more than either agent alone. Such combination therapy may be beneficial clinically by potentiating cardioprotection after coronary reperfusion at doses far below those producing vasodilatation or side effects.

Quantification of cardiomyocyte contraction is usually obtained by measuring globally cell shortening from the displacement of cell extremities. We developed a correlationbased optical flow method, which correlates the whole-cell temporal pattern with a precise quantification of the intracellular strain wave at the sarcomeres level. A two-dimensional image correlation analysis of cardiomyocytes phase-contrast images was developed to extract local cell deformations from videomicroscopy time-lapse sequences. Test images, synthesized from known intensity displacement fields, were first used to validate the method. Intracellular strain fields were then computed from videomicroscopy time-lapse sequences of single adult and neonatal cardiomyocytes. The propagation of the sarcomeres contraction-relaxation wave during cell contraction has been successfully quantified. The time-varying patterns of intracellular displacement were obtained accurately, even when cardiomyocyte bending occurred in pace with contraction. Interestingly, the characterization of the successive 2D displacement fields show a direct quantification of the variation with time of intracellular strains anywhere in the cell. The proposed method enables a quantitative analysis of cardiomyocyte contraction without requiring wave tracking with the use of fluorescent calcium probes. Thus, our algorithmic approach provides a fast and efficient tool for analyzing the correlation between global cell dynamical behavior and mechanosensitive intracellular processes.

(99m)Tc-RAFT-RGD allowed the in vivo imaging of alpha(v)beta(3) integrin tumour expression.

99m Tc-cAbVCAM1-5, a single-domain antibody fragment directed against mouse or human vascular cell adhesion molecule 1 (VCAM-1), recently has been proposed as a new imaging agent for the detection of inflamed atherosclerotic lesions. Indeed, in a mouse model of atherosclerosis, 99m Tc-cAbVCAM1-5 specifically bound to VCAM-1-positive lesions, thereby allowing their identification on SPECT images. The purpose of the present study was to investigate 99m TccAbVCAM1-5 imaging sensitivity using a reference statin therapy. Methods: Thirty apolipoprotein E-deficient mice were fed a westerntype diet. First, the relationship between the level of VCAM-1 expression and 99m Tc-cAbVCAM1-5 uptake was evaluated in 18 mice using immunohistochemistry and autoradiography. Second, longitudinal SPECT/CT imaging was performed on control (n 5 9) or atorvastatintreated mice (0.01% w/w, n 5 9). Results: 99m Tc-cAbVCAM1-5 uptake in atherosclerotic lesions correlated with the level of VCAM-1 expression (P , 0.05). Atorvastatin exerted significant antiatherogenic effects, and 99m Tc-cAbVCAM1-5 lesion uptake was significantly reduced in 35-wk-old atorvastatin-treated mice, as indicated by ex vivo γ-well counting and autoradiography (P , 0.05). SPECT imaging quantification based on contrast-enhanced CT was reproducible (interexperimenter intraclass correlation coefficient, 0.97; intraexperimenter intraclass correlation coefficient, 0.90), and yielded results that were highly correlated with tracer biodistribution (r 5 0.83; P , 0.0001). Therefore, reduced 99m Tc-cAbVCAM1-5 uptake in atorvastatin-treated mice was successfully monitored noninvasively by SPECT/CT imaging (0.87 ± 0.06 vs. 1.11 ± 0.09 percentage injected dose per cubic centimeter in control group, P , 0.05). Conclusion: 99m TccAbVCAM1-5 imaging allowed the specific, sensitive, and reproducible quantification of VCAM-1 expression in mouse atherosclerotic lesions. 99m Tc-cAbVCAM1-5 therefore exhibits suitable characteristics for the evaluation of novel antiatherogenic agents.

Background-Adenosine (Ado) and dipyridamole are alternatives to exercise stress for myocardial perfusion imaging.Though generally safe, side effects frequently occur that cause patient discomfort and sometimes lead to premature termination of the study or require aminophylline administration. Recently, a new class of A 2A Ado receptor agonists was synthesized. ATL193 and ATL146e are 2-propynylcyclohexyl-5Ј-N-ethylcarboxamido derivatives of Ado. The study goals were to evaluate the potency and selectivity of these new compounds on recombinant canine Ado receptors and to evaluate their hemodynamic properties in dogs to assess their usefulness as vasodilators for myocardial perfusion imaging. Methods and Results-In assays of recombinant canine Ado receptors, ATL-193 and ATL-146e were highly selective for the A 2A over the A 1 and A 3 receptors and were more potent than MRE-0470 and CGS-21680. In 16 anesthetized dogs, the agonists were administered by infusion (ATL-193; nϭ7 normal) or bolus injection (ATL-146e; nϭ9 critical left anterior descending coronary artery stenosis), and hemodynamic responses were compared with those of Ado. Both agonists produced dose-dependent coronary flow (CF) elevation without provoking the hypotension observed with Ado.After an ATL-146e bolus, the CF increase was sustained for several minutes, providing ample time for injection and myocardial uptake of 99m Tc-sestamibi, and CF returned to baseline within 20 minutes. The CF increase was completely blocked by the selective A 2A antagonist ZM241385 (3 g · kg Ϫ1 · min Ϫ1 ). Conclusions-ATL-193 and ATL-146e are highly potent and selective Ado A 2A receptor agonists with excellent potential for use as vasodilators for myocardial perfusion imaging. An important advantage of ATL-146e is the ability to administer it by bolus injection.