In this study, we showed that MPO activity in rabbit atherosclerotic plaques, and thus biologically relevant active inflammation, can be detected non-invasively using a clinical MR scanner by employing the MPO-activatable agent MPO(Gd). MPO rich areas are selectively enhanced by MPO(Gd) and easily identified 120 minutes post-administration. We verified that the foci of increased intensity on MPO(Gd) imaging co-localized and correlated with MPO-rich areas infiltrated by macrophages on histopathological evaluations. Biochemical assays showed that atherosclerotic plaques possessed elevated MPO activity compared to normal arterial walls, and that MPO activity correlated positively with plaque size. The results demonstrate that in vivo MPO activity is well associated with atherosclerotic plaque development and progression.
Our animal model develops arterial plaques that exhibit several plaque features that have been described as markers of plaque vulnerability in human plaques, including neovascularization and extensive macrophage infiltration 2, 30
. Interestingly, some areas within the plaques were rich in macrophages but not in MPO ( and S3
), which agrees with the concept of distinct macrophage phenotypes within plaques 17
. This was supported by the strong correlation of MPO(Gd) imaging to MPO immunostaining, but not macrophage staining, since areas containing MPO-poor macrophages should not be highlighted by our agent (). There was also a mild positive correlation of MPO(Gd) imaging with lipid histopathology but not with collagen histopathology (), consistent with the concept that lipid cores are associated with more advanced unstable plaques and that many of the macrophages that produced MPO were rich in lipid (foam cells). These findings underscore the importance of our functional approach. Furthermore, the plaques in our rabbit model expressed substantially lower amounts of MPO (mean 7.7 U/mg protein) than did human atherosclerotic tissue (mean 251 U/mg protein) 16
. While underscoring some differences between rabbit and human plaques, this also has important implications for the translation of this agent for imaging human plaques, since the increased MPO content of human plaques should result in substantially greater signal intensity at sites of MPO(Gd) activation, which could allow lower doses of the agent to be used as well as increasing the sensitivity for detecting plaque vulnerability at the earliest stages of development.
Previous enzyme-sensitive MRI agents are based on a cleavage mechanism to remove masking groups that limit water access 31
, albumin binding groups 32
, or solubility 33
. These studies represented significant advances in imaging agent design. However, to date these prototype agents have not been shown to be effective in vivo
without significant manipulations of the test animals (which had been limited to invertebrates and small mammals), and unlike MPO(Gd), cannot be administered intravenously, important for clinical translation. MPO(Gd) achieves signal amplification by enzymatic addition instead of cleavage. In a mouse model of myocardial infarction treated with atorvastatin, we found that MPO(Gd) was sufficiently sensitive to detect a decrease in MPO activity and inflammation 23
. Furthermore, MPO(Gd) discriminated between wild type mice with full MPO expression, MPO heterozygous mice with intermediate MPO expression, and MPO knockout mice with no MPO expression. As MPO is highly conserved across mammalian species 34
, these and the results in this study confirm high sensitivity and specificity of the agent for MPO activity.
In summary, key advantages of this molecular technology lie in enabling clinical MRI scanning and T1-weighted sequences to identify pathology noninvasively and to localize and track harmful oxidative reactions in atherosclerotic lesions. An additional advantage is the short readout delay between injection and imaging (90-120 minutes). Our study provides initial proof-of-priniciple for a new, more specific imaging approach of inflammation in atherosclerosis by imaging macrophage function and the activity of an effector enzyme, and thus is of direct biological relevance. Unlike measuring the presence of phagocytes, measuring MPO activity in plaques is likely to have greater predictive value on the risk of plaque rupture. This technology thus can localize plaques with significant active inflammation prior to devastating thromboembolic events. Consequently, it could change clinical standards by enabling earlier diagnosis and improved risk-stratification as well as allowing the ability to track the effects of timely, patient-specific interventions.