Abstract :
[en] Introduction. 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 [1]. In this context, phosphatidylserine exposure on the outer leaflet of the cell membrane is one of the earliest detectable molecular events in apoptosis. 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 MRI. A phosphatidylserine-specific peptide identified by phage display was thus used to design an MRI contrast agent (CA) that was evaluated as a potential in vivo reporter of apoptotic cells.
Methods. A library of linear 6-mer random peptides (fused to the pIII protein of M13 bacteriophage) was screened in vitro against immobilized phosphatidylserine. Phage DNA was isolated and sequenced by Sanger method. The affinity of peptides for phosphatidylserine was evaluated by ELISA, using the Kd and IC50 as criteria of peptide selection. The phosphatidylserine-specific peptide and its scrambled homologous were attached to a linker (8-amino-3,6-dioxaoctanoyl) and conjugated to DTPA-isothiocyanate. The products were purified by dialysis and by column chromatography, and complexed with gadolinium chloride. After its 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.
Results. 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, which reminds the function of annexins. Alignment with molecules involved in apoptosis, i.e. Fas antigen ligand and apoptosis associated tyrosine-kinase, provides a direct correlation between peptide selection and utility. The in vivo MRI studies performed at 4.7T suggest that the majority of the increased liver or aorta localization of the new phosphatidylserine-targeted CA is the result of a specific interaction with apoptotic cells, providing proof of concept that apoptosis-related pathologies could be diagnosed by MRI with a low-molecular weight paramagnetic agent.
Conclusion. The new CA could have a 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.
References
1. Tabas I. Apoptosis and plaque destabilization in atherosclerosis: the role of macrophage apoptosis induced by cholesterol. Cell Death Differ. 2004, 11, S12-S16.
