Faculty Bibliography

Appreciation of the molecular and cellular processes of atherosclerosis, thrombosis, and vascular inflammation has identified new targets for imaging. The common goals of molecular imaging approaches are to accelerate and refine diagnosis, provide insights that reveal disease diversity, guide specific therapies, and monitor the effects of those therapies. Here we undertake a comparative analysis of imaging modalities that have been used in this disease area. We consider the elements of contrast agents, emphasizing how an understanding of the biology of atherosclerosis and its complications can inform optimal design. We address the potential and limitations of current contrast approaches in respect of translation to clinically usable agents and speculate on future applications.

Determining arterial macrophage expression is an important goal in the molecular imaging of atherosclerosis. Here, we compare the efficacy of two synthetic, high density lipoprotein (HDL) based contrast agents for magnetic resonance imaging (MRI) of macrophage burden. Each form of HDL was labeled with gadolinium and rhodamine to allow MRI and fluorescence microscopy. Either the 37 or 18 amino acid peptide replaced the apolipoprotein A-I in these agents, which were termed 37pA-Gd or 18A-Gd. The diameters of 37pA-Gd and 18A-Gd are 7.6 and 8.0 nm, respectively, while the longitudinal relaxivities are 9.8 and 10.0 (mM s)(-1). 37pA has better lipid binding properties. In vitro tests with J774A.1 macrophages proved the particles possessed the functionality of HDL by eliciting cholesterol efflux and were taken up in a receptor-like fashion by the cells. Both agents produced enhancements in atherosclerotic plaques of apolipoprotein E knockout mice of approximately 90% (n = 7 per agent) and are macrophage specific as evidenced by confocal microscopy on aortic sections. The half-lives of 37pA-Gd and 18A-Gd are 2.6 and 2.1 h, respectively. Despite the more favorable lipid interactions of 37pA, both agents gave similar, excellent contrast for the detection of atherosclerotic macrophages using MRI.

There is an ongoing desire to produce high-relaxivity, Gd-based magnetic resonance imaging (MRI) contrast agents. These may allow for lower doses to be used, which is especially important in view of the current safety concerns surrounding Gd in patients. Here we report the synthesis of a high-relaxivity MRI contrast agent, by incorporating Gd-chelating lipids that coordinate two water molecules into high-density lipoprotein (q = 2 HDL). We compared the properties of q = 2 HDL with those of an analogous HDL particle labeled with Gd-chelating lipids that coordinate only one water molecule (q = 1 HDL). We found that the q = 2 HDL possessed an elevated r(1) of 41 mM(-1) s(-1) compared to 9 mM(-1) s(-1) for q = 1 HDL at 20 MHz, but the q = 2 HDL exhibited high R(2)* values at high fields, precluding imaging above 128 MHz. While carrying out this investigation we observed that enlarged, disrupted particles were formed when the synthesis was carried out above the lipid critical micelle concentration (cmc), indicating the importance of synthesis below the cmc when modifying lipoproteins in this manner. The high relaxivity of q = 2 HDL means it will be an efficacious contrast agent for future MR imaging studies

PURPOSE: To evaluate the capability of P947, a magnetic resonance (MR) imaging contrast agent that molecularly targets matrix metalloproteinases (MMPs), to aid detection and imaging of MMPs in atherosclerotic lesions in vivo; its specificity compared with that of P1135; expression and distribution of MMPs in atherosclerotic vessels; and in vivo distribution and molecular localization of fluorescent europium (Eu) P947. MATERIALS AND METHODS: The Animal Care and Use Committee approved all experiments. P947 was synthesized by attaching a gadolinium chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) to a peptide that specifically binds MMPs. Scrambled form of P947 (P1135) was synthesized by replacing the targeting moiety of P947 with a scrambled peptide lacking the ability to bind MMPs. P947, P1135, and gadoterate meglumine were injected into atherosclerotic apolipoprotein E-deficient and wild-type mice. The aortic MR imaging enhancement produced by the contrast agents was measured at different times and was compared by using one-way analysis of variance. MMP expression was investigated in the aortas by using MMP immunostaining and in situ MMP zymography. A fluorescent form of P947 (Eu-P947) was synthesized to compare the in vivo distribution of the contrast agent (Eu-P947) with specific MMP immunofluorescent staining. RESULTS: MMP-targeted P947 facilitated a 93% increase (P < .001) in MR image signal intensity (contrast-to-noise ratio [CNR], 17.7 compared with 7.7; P < .001) of atherosclerotic lesions in vivo. Nontargeted P1135 (scrambled P947) provided 33% MR image enhancement (CNR, 10.8), whereas gadoterate meglumine provided 5% (CNR, 6.9). Confocal laser scanning microscopy demonstrated colocalization between fluorescent Eu-P947 and MMPs in atherosclerotic plaques. Eu-P947 was particularly present in the fibrous cap region of plaques. CONCLUSION: P947 improved MR imaging for atherosclerosis through MMP-specific targeting. The results were validated and provide support for further assessment of P947 as a potential tool for the identification of unstable atherosclerosis.