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

Abstract

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.

Development of a Magnetic Resonance Imaging Protocol for the Characterization of Atherosclerotic Plaque by Using Vascular Cell Adhesion Molecule-1 and Apoptosis-Targeted Ultrasmall Superparamagnetic Iron Oxide Derivatives

“…17 Human umbilical vein endothelial cells (HUVEC) were stimulated with tumor necrosis factor- (Alexis Biochemicals, Zandhoven, Belgium). 16 The cells were then incubated with USPIO derivatives at an iron concentration of 4, 3, and 2 mmol/L.…”

“…16,17 Before harvesting the aorta, the mice were euthanized by a lethal dose of Nembutal and the blood was removed by transcardial perfusion with 10 mL of PBS; this procedure prevents the endogenous iron staining by the Perls23,3-diaminobenzidine (DAB) protocol. Endogenous peroxidases, nonspecific epitopes, and endogenous biotin were blocked as described previously.…”

“…Endogenous peroxidases, nonspecific epitopes, and endogenous biotin were blocked as described previously. 16,17 For immunohistochemistry of VCAM-1 (biomarker of inflammation and of angiogenesis) and of caspase-3 (biomarker of apoptosis), the sections were incubated with rabbit anti-VCAM-1 polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) or with rabbit anti-caspase-3 polyclonal antibody (Abcam, Cambridge, UK), followed by biotinylated goat antirabbit IgG (Vector Labconsult) and by the Vectastain ABC kit. Mac-1 (marker of macrophages) was immunolabeled with rat monoclonal antimouse MAC-1 (CD11b) antibody (AbD Serotec, Düsseldorf, Germany), followed by biotinylated rabbit antirat IgG (Vector Labconsult) and by the ABC kit.…”

“…15 During our previous studies, 2 peptides were identified by phage display and used to target the vascular cell adhesion molecule-1 (VCAM-1), as a biomarker of inflammation (targeted with a cyclic heptapeptide: R832), 16 and phosphatidylserine, as a biomarker of apoptosis (targeted with a linear hexapeptide: R826). 17 After conjugating them to paramagnetic contrast agents, the molecular imaging probes were validated on apolipoprotein E (ApoE) knockout (KO) mice by magnetic resonance imaging (MRI). However, the paramagnetic probes are known for their low relaxivity (ie, MRI efficacy, expressed as r 1 and r 2 , which is defined as the increase of relaxation rate [R 1 51/T 1 ; R 2 51/T 2 ] produced by 1 mmol/L of contrast agent [expressed in s/mmol per L]), which represents a serious drawback especially for molecular imaging of scarce targeted biomarkers in the diseased tissues.…”

Targeting of cell death and neuroinflammation with peptide-linked iron oxide nanoparticles and Gd-DTPA in a mouse model of Parkinson's disease

“…These specific contrast agents are linked to peptides that show affinity for special molecular targets abundant in or specific to the injured areas of some pathology. In this work, two peptides selected by the phage display method were linked to iron oxide nanoparticles and Gd-DTPA (gadolinium-diethylenetriamine pentaacetic acid): 2C-peptide for MRI detection of inflammation [21] and R826-peptide for apoptosis detection [22] in a mouse model of PD.…”