Laurent M. Riou

In Vivo Molecular Imaging of Atherosclerotic Lesions in ApoE−/− Mice Using VCAM-1–Specific, 99mTc-Labeled Peptidic Sequences

Vascular cell adhesion molecule 1 (VCAM-1) plays a major role in the chronic inflammatory processes involved in vulnerable atherosclerotic plaque development. We previously showed that the 99mTc-labeled major histocompatibility complex 1–derived peptide B2702p bound specifically to VCAM-1 and allowed the ex vivo imaging of atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits. However, B2702p target-to-background ratio was suboptimal for the in vivo imaging of VCAM-1 expression in atherosclerotic lesions. To improve the target-to-background ratio, 20 derivatives of B2702p (B2702p1–B2702p20) were synthesized using the alanine scan methodology. We hypothesized that 99mTc-radiolabeled B2702p derivatives might allow the molecular imaging of VCAM-1 expression in an experimental model of atherosclerosis. Methods: A mouse model of focal atherosclerotic plaque development induced by left carotid artery ligation in apolipoprotein E double-knockout (ApoE−/−) mice was used (n = 82). 99mTc-B2702p and 99mTc-B2702p1–99mTc-B2702p20 were injected intravenously in anesthetized animals 3 wk after the ligation. Whole-body planar imaging was performed for 3 h. SPECT imaging of 6 additional ligated ApoE−/− mice was also performed with 99mTc-B2702p1. The animals were then euthanized, and the biodistribution of 99mTc-labeled peptides was evaluated by γ-well counting of excised organs. Expression of VCAM-1 in the ligated and contralateral carotid arteries was evaluated by immunohistology. Results: Robust VCAM-1 immunostaining was observed in the left carotid atherosclerotic lesions as a consequence of artery ligation, whereas no VCAM-1 expression was detected in the contralateral carotid artery. Among all evaluated peptides, 99mTc-B2702p1 exhibited the most favorable properties. By γ-well counting, there was a significant 2.0-fold increase in the 99mTc-B2702p1 left-to-right carotid artery activity ratio (2.6 ± 0.6) and a 3.4-fold increase in the left carotid-to-blood activity ratio (1.4 ± 0.4) in comparison to 99mTc-B2702p (1.3 ± 0.2 and 0.4 ± 0.1, respectively, P < 0.05 for both comparisons). Similarly, planar image quantification indicated a higher left-to-right carotid activity ratio in 99mTc-B2702p1– than in 99mTc-B2702p–injected mice (1.2 ± 0.1 vs. 1.0 ± 0.0, respectively, P < 0.05). Finally, a significantly higher 99mTc-B2702p1 activity in the left than in the right carotid artery was observed by SPECT imaging (2.2 ± 0.4 vs. 1.4 ± 0.3 cpm/mm2/injected dose, respectively, P < 0.05). Conclusion: 99mTc-B2702p1 is a potentially useful radiotracer for the in vivo molecular imaging of VCAM-1 expression in atherosclerotic plaques.

99mTc-cAbVCAM1-5 Imaging Is a Sensitive and Reproducible Tool for the Detection of Inflamed Atherosclerotic Lesions in Mice

99mTc-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, 99mTc-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 99mTc-cAbVCAM1-5 imaging sensitivity using a reference statin therapy. Methods: Thirty apolipoprotein E–deficient mice were fed a western-type diet. First, the relationship between the level of VCAM-1 expression and 99mTc-cAbVCAM1-5 uptake was evaluated in 18 mice using immunohistochemistry and autoradiography. Second, longitudinal SPECT/CT imaging was performed on control (n = 9) or atorvastatin-treated mice (0.01% w/w, n = 9). Results: 99mTc-cAbVCAM1-5 uptake in atherosclerotic lesions correlated with the level of VCAM-1 expression (P < 0.05). Atorvastatin exerted significant antiatherogenic effects, and 99mTc-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 = 0.83; P < 0.0001). Therefore, reduced 99mTc-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: 99mTc-cAbVCAM1-5 imaging allowed the specific, sensitive, and reproducible quantification of VCAM-1 expression in mouse atherosclerotic lesions. 99mTc-cAbVCAM1-5 therefore exhibits suitable characteristics for the evaluation of novel antiatherogenic agents.

Effects of mechanical properties and atherosclerotic artery size on biomechanical plaque disruption - mouse vs. human.

Mouse models of atherosclerosis are extensively being used to study the mechanisms of atherosclerotic plaque development and the results are frequently extrapolated to humans. However, major differences have been described between murine and human atherosclerotic lesions and the determination of similarities and differences between these species has been largely addressed recently. This study takes over and extends previous studies performed by our group and related to the biomechanical characterization of both mouse and human atherosclerotic lesions. Its main objective was to determine the distribution and amplitude of mechanical stresses including peak cap stress (PCS) in aortic vessels from atherosclerotic apoE(-/-) mice, in order to evaluate whether such biomechanical data would be in accordance with the previously suggested lack of plaque rupture in this model. Successful finite element analysis was performed from the zero-stress configuration of aortic arch sections and mainly indicated (1) the modest role of atherosclerotic lesions in the observed increase in residual parietal stresses in apoE(-/-) mouse vessels and (2) the low amplitude of murine PCS as compared to humans. Overall, the results from the present study support the hypothesis that murine biomechanical properties and artery size confer less propensity to rupture for mouse lesions in comparison with those of humans.

Evaluation of anti-atherogenic properties of ezetimibe using 3H-labeled LDL and 99mTc-cAbVCAM1-5 SPECT imaging in ApoE-/- mice fed a paigen diet

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E−/− mice. Methods: Apolipoprotein E−/− mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (n = 15), we intravenously injected 3H-cholesteryl oleate–labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (n = 20), we used the imaging agent 99mTc-cAbVCAM1–5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (n = 21), we compared VCAM-1 expression with 99mTc-cAbVCAM1–5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL–cholesteryl ester plasma disappearance rate (P < 0.001 vs. control) after 3H-cholesteryl oleate–labeled LDL injection. At 96 h after injection, the hepatic fraction of 3H-tracer was 61% lower in mice treated with ezetimibe (P < 0.001). Meanwhile, LDL-derived 3H-cholesterol excretion in the feces was 107% higher (P < 0.001). The antiatherogenic effect of ezetimibe monitored by 99mTc-cAbVCAM1–5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with 99mTc-cAbVCAM1–5 uptake (r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.