Molecular and cellular targets of the MRI contrast agent P947 for atherosclerosis imaging

Abstract

P947 (DOTA-Gd-peptide) was recently identified as an MRI contrast agent for the detection and characterization of the matrix metalloproteinases (MMP)-rich atherosclerotic plaques. Because this product displays a broad spectrum affinity for the MMP family, we hypothesized that it may also recognize other metalloproteinases overactivated in vulnerable atherosclerotic plaques. Therefore, this study aimed at describing, at the molecular and cellular level, the interactions between P947 and proteases of atherosclerotic plaques. Fluorimetric assays were used to measure the in vitro affinity of P947 toward recombinant and purified MMPs, angiotensin-converting enzyme (ACE), endothelin-converting enzyme (ECE-1), neutral endopeptidase (NEP), and both aminopeptidases A and N (APA and APN). Using similar fluorimetric assays associated with specific substrates, enzymatic activities were measured in vulnerable and stable plaques collected from human atherosclerotic carotid arteries. Ex vivo affinity of P947 for metalloproteinases in vulnerable lesions was subsequently determined. Interaction between P947 and major cell types present in atherosclerotic plaques was also investigated in different cell lines: PMA-1-differentiated THP-1 (macrophage), Ox-LDL-treated THP-1 (foam cell), Jurkat cell line (lymphocyte), and human umbilical vein endothelial cell (HUVEC, endothelial cell). Molecular targeting of P947 was confirmed by fluorimetry, ICP-MS, and in vitro MRI approaches. Potential application of P947 for detecting atherosclerotic plaques by in vivo MRI was tested in a rabbit model of atherosclerosis. In vitro, P947 displayed affinities for purified MMPs, ACE, ECE-1, NEP, APA, and APN in the micromolar range. Interestingly, MMPs, ACE, and APN exhibited higher activities in vulnerable plaques from human atherosclerotic carotid samples, as compared to stable plaques. ECE-1, NEP, and APA had either no activity or the same low activity in both vulnerable and stable plaques. P947 showed micromolar affinities for MMPs, ACE, and APN secreted by plaque samples. Moreover, P947 bound to THP-1 macrophages and THP-1 foam cells in a concentration-dependent manner and with a higher intensity than the control contrast agents DOTA-Gd or P1135 (DOTA-Gd coupled to a scrambled peptide). In THP-1 macrophages, P947 inhibited largely (70%) and almost completely (95%) MMP and APN activities, respectively, which strongly suggested an MMP- and APN-dependent binding of P947 to these cells. This enzyme-specific binding was confirmed with in vitro MRI. Indeed, the T1 value of THP-1 cells decreased from 2.094 s (macrophages w/o P947) to 2.004 s (macrophages with 1 mM of P947). In addition, the Gd content measured by ICP-MS was 11.01 ± 1.05 fg Gd/macrophage when cells were incubated in the presence of P947 and only 5.18 ± 0.43 fg Gd/macrophage with the control product P1135. The difference of Gd concentration between both contrast agents corresponded to a specific accumulation of 5.83 fg Gd/cell, which may be detected by MRI. MR imaging in the atherosclerosis rabbit model showed enhancement of the aortic wall after P947 injection with a significant increase of CNR values from 0.21 ± 0.02 (before injection) to 0.37 ± 0.07 (after injection), demonstrating the efficacy of the contrast agent to detect the atherosclerotic plaques in vivo. Taken together, these data suggest that P947 may be an interesting contrast agent for in vivo molecular MR imaging of MMPs, ACE, and APN activities present in vulnerable atherosclerotic plaques.