Peptidic Targeting of Phosphatidylserine for the MRI Detection of Apoptosis in Atherosclerotic Plaques

• Published in: Molecular pharmaceutics 2009-12, Vol.6 (6), p.1903-1919
• Main Authors: Burtea, Carmen, Laurent, Sophie, Lancelot, Eric, Ballet, Sébastien, Murariu, Oltea, Rousseaux, Olivier, Port, Marc, Vander Elst, Luce, Corot, Claire, Muller, Robert N
• Format: Article
• Language: English
• Subjects: Animals; Apolipoproteins E - genetics; Apoptosis - physiology; Atherosclerosis - metabolism; Atherosclerosis - pathology; Caspase 3 - analysis; Cells, Cultured; Contrast Media - chemistry; Female; Immunohistochemistry; Liver - pathology; Magnetic Resonance Imaging - methods; Mice; Mice, Inbred BALB C; Mice, Knockout; Models, Theoretical; Molecular Structure; Peptides - chemistry; Phosphatidylserines - chemistry; Phosphatidylserines - metabolism
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/mp900106m

Molecular and cellular imaging of atherosclerosis has garnered more interest at the beginning of the 21st century, with aims to image in vivo biological properties of plaque lesions. Apoptosis seems an attractive target for the diagnosis of vulnerable atherosclerotic plaques prone to a thrombotic event. The aim of the present work was to screen for apoptosis peptide binders by phage display with the final purpose to detect apoptotic cells in atherosclerotic plaques by magnetic resonance imaging (MRI). A phosphatidylserine-specific peptide identified by phage display was thus used to design an MRI contrast agent (CA), which was evaluated as a potential in vivo reporter of apoptotic cells. A library of linear 6-mer random peptides was screened in vitro against immobilized phosphatidylserine. Phage DNA was isolated and sequenced, and the affinity of peptides for phosphatidylserine was evaluated by enzyme-linked immunosorbent assay. The phosphatidylserine-specific peptide and its scrambled homologue were attached to a linker and conjugated to DTPA-isothiocyanate. The products were purified by dialysis and by column chromatography and complexed with gadolinium chloride. After their evaluation using apoptotic cells and a mouse model of liver apoptosis, the phosphatidylserine-targeted CA was used to image atherosclerotic lesions on ApoE−/− transgenic mice. Apoptotic cells were detected on liver and aorta specimens by the immunostaining of phosphatidylserine and of active caspase-3. Sequencing of the phage genome highlighted nine different peptides. Their alignment with amino acid sequences of relevant proteins revealed a frequent homology with Ca2+ channels, reminiscent of the function of annexins. Alignment with molecules involved in apoptosis provides a direct correlation between peptide selection and utility. The in vivo MRI studies performed at 4.7 T provide proof of concept that apoptosis-related pathologies could be diagnosed by MRI with a low molecular weight paramagnetic agent. The new CA could have real potential in the diagnosis and therapy monitoring of atherosclerotic disease and of other apoptosis-associated pathologies, such as cancer, ischemia, chronic inflammation, autoimmune disorders, transplant rejection, neurodegenerative disorders, and diabetes mellitus. The phage display-derived peptide could also play a potential therapeutic role through anticoagulant activity by mimicking the role of annexin V, the endogenous ligand of phosphatidylserine.