Vardan Amirbekian, M.D., 2004-06 Sarnoff Fellow Department of Radiology

1. Magnetic Resonance Imaging (MRI) of Atherosclerosis Using Molecularly Targeted Nano-scale Gadolinium Immunomicelles. Vardan Amirbekian, M.D., 2004-06 Sarnoff Fellow Department of Radiology Brigham and Women’s Hospital – Harvard Medical School

2. Current Contrast-Enhanced MRI Limited Plaque Activity Information Gd-chelate enhanced -non specific Targeted enhanced contrast agent - specific interaction with plaque Sirol M; Fayad ZA et al.

3. monocyte cholesterol disposal In liver VLA-4 HDL MCP-1 LCAT VCAM-1 ICAM-1 M-CSF apoAI endothelium MSR-A LDL ABCA-1 Ox-LDL CD-36 Freecholesterol Choudhury R et al. Nature Cardiovascular Meidine 2005 SR-B1 NCEH ACAT-1 Cholesteryl ester exit to lymphatic system macrophage foam cell

4. Gd-Immunomicelles • Conjugation of Gd(III) to hydrophillic side chains (corona) • Self-assemble • Amphilic components • Hydrophobic lipid core with polar surface Diameter~ 80-120 nm diameter 4,000 Gd/particle r1=~20 s-1mM-1 (63 MHz in H20) • Incorporation of ligands that specifically target a molecule of interest (the ligand can be a Antibody, peptide and any other structure with specific affinity for a molecule of interest). • Conjugation of Gd(III) to hydrophillic side chains

5. Hypothesis Targeting Gadolinium-containing MRI contrast-agents, such as immunomicelles, to the Macrophage Scavenger Receptor (MSR) will facilitate detection of atherosclerosis in Apo E knockout mice and furthermore may provide information about macrophage plaque content and plaque activity. The goals of the current study were: (i) to evaluate the relationship between macrophage content of plaques and the changes seen in MRI signal intensity using immunomicelles targeted to MSR; (ii) to examine the effect of MSR inhibition on immunomicelles-mediated MRI enhancement of atherosclerosis.

6. Experimental Design - Methods • Micelles, immunomicelles, and standard (Gd-DTPA) paramagnetic contrast agents were tested in ApoE KO mice. • Mice were imaged at baseline with a 9.4T MR system (Bruker) using a high-spatial resolution sequence (98µmx98µmx500µm). • Mice were then injected with either micelles, immunomicelles, or standard. • Mice were then imaged at 1-hour, 24-hrs, 48-hrs and 72-hrs and 1-week post-contrast-injection. • Mice were all sacrificed at the end of the imaging period. Afterwards various types of analyses were performed including standard pathology, histology, and immunohistopathology.

7. In Vivo Aortic Atherosclerotic Plaque in ApoE-KO Mouse Gd-Immunomicelles Targeted to MSR ApoE-KO WT Pre-contrast 60 min. post 24hr-post Amirbekian, V. et al. PNAS 2007

8. Amirbekian, V. et al. PNAS 2007

9. CD68 Macrophage Immunostaining of the Plaques Imaged in vivo with Immunomicelles Macrophages were counted per HPF and these values were plotted against the %NER observed by Immunomicelles-mediated MRI at the same anatomic level. Amirbekian, V. et al. PNAS 2007

10. Amirbekian, V. et al. PNAS 2007

11. Macrophages and MSR-Targeted Immunomicelles in Atherosclerotic Plaque DAPI Nuclei Fluorescent Immunomicelles CD68 Macrophages Overlay Amirbekian, V. et al. PNAS 2007

12. Conclusions • The current in vivo study shows that, using MRI, immunomicelles targeted to macrophages may aid in the non-invasive detection of atherosclerotic plaque. • There appears to be a direct relationship between macrophage content of plaques and the changes seen in MRI signal intensity using immunomicelles targeted to MSR. • Immunomicelles may prove useful in detection of high-macrophage density typical of high-risk plaques. They may also provide valuable information about plaque activity.

13. Hypothesis Immunomicelles targeting the macrophage scavenger receptor-B (CD36) will specifically improve ex-vivo MR detection and characterization of human aortic atherosclerosis.

14. Experimental Design - Methods • Gd-containing micelles, anti-CD36 immunomicelles and Fc-micelles were created. • Macrophages were incubated with fluorescent micelles and immunomicelles to determine uptake via confocal microscopy and inductively coupled plasma mass spectroscopy (ICP-MS) was performed to quantify Gd uptake. • Human aortic specimens with moderate to severe atherosclerosis were harvested at autopsy. Using a 1.5 T Siemens clinical scanner, T1, T2, and PDW 3-dimensional scans were performed and post-contrast scans were repeated after 24 h incubation. • T1 analysis and cluster analysis were performed comparing immunohistopathology with MR images. • P-values<0.05 were considered significant.

15. Immunomicelles – macrophage uptake in vitro and human atherosclerotic uptake ex vivo

16. MRI Results in Human Atherosclerosis

17. Co-localization of Immunomicelles and Macrophages

18. Brigham and Women’s Hospital Harvard Medical School Dept. of Radiology Dr. Barbara N. Weissman Dr. Steven E. Seltzer Dr. Frank J. Rybicki Dr. Salvatore G. Viscomi Bruce Boynton Jenna Hastings Mount Sinai School of Medicine Institute for Translational and Molecular Imaging Dr. Zahi A. Fayad Smbat Amirbekian, BS. Dr. Fabien Hyafil Dr. Esad Vucic Dr. Marc Sirol Dr. Juan G.S. Aguinaldo Dr. Karen C. Briley-Saebo Dr. Edward Fisher Depts. of Radiology & Cardiology at Mount Sinai I am Grateful to: Johns Hopkins University School of Medicine Dr. Donna Magid Dr. Charles Lowenstein