Design and Validation of a Specific Scavenger Receptor Class AI Binding Peptide for Targeting the Inflammatory Atherosclerotic Plaque

Abstract

Objective-Scavenger receptor A (SR-A) is abundantly expressed by macrophage and plays a critical role in foam cell formation and atherogenesis. In search of selective SR-AI antagonists, we have used affinity selection of a phage displayed peptide library on the synthetic extracellular domain of SR-AI. Methods and Results-Phage selection led to an almost 1,000-fold enrichment of SR-AI binding phage, which bound avidly to human THP-1 cells. A 15-mer corresponding to the peptide insert of the major SR-AI binding phage (PP1) displaced phage binding to SR-AI. Peptides, docked to a streptavidin scaffold, were effectively internalized by macrophages in an SR-AI-dependent manner. The enriched phage pool and streptavidin bound PP1 exhibited marked uptake by hepatic macrophages in mice. Importantly, PP1 significantly increased streptavidin as well as particulate accumulation in advanced aortic plaques, and in particular intraplaque macrophage, of apolipoprotein E Ϫ/Ϫ mice. 2,6 and can exist in 1 of 3 isoforms (SR-AI, SR-AII, and SR-AIII). Conclusion7,8 As a pattern recognition receptor, it displays a very broad substrate profile, internalizing modified low-density lipoprotein, polynucleotides (eg, polyinosinic and polyguanylic acid), polysaccharides (eg, fucoidan), and lipopolysaccharides.9-11 SR-A was found to be abundantly expressed by macrophages, foam cells, and smooth muscle cells 12 in atherosclerotic lesions but not in the normal vessel wall.13,14 A causal role of SR-A in atherogenesis was first established by Suzuki et al, 4 showing reduced lesion formation in SR-A-deficient apolipoprotein E (ApoE) Ϫ/Ϫ , a notion confirmed in further studies. 15The marked expression of SR-AI by macrophages in plaques-especially vulnerable plaques-and the efficient endocytosis of its substrates by SR-AI render it not only an interesting candidate for therapeutic intervention in atherosclerosis but also for targeted drug delivery and imaging approaches. 16 Application of selective SR-AI ligands as homing device for contrast agents may therefore allow molecular imaging of atherosclerotic plaques in general but also give insight into their actual composition and stability. Illustrative, macrophagetargeted molecular magnetic resonance imaging of atherosclerotic plaques has already been shown for immunomicelles equipped with antibodies for macrophage scavenger receptor 17 or for oxidation specific epitopes. 18The bulky and chemically complex nature of its macromolecular substrates make ligand design for SR-AI rather unsuccessful, and only a single report was published on a synthetic, moderately selective SR-A antagonist. 19 In this study, we describe the unbiased design of SR-AI antagonists involving the use of phage display on a synthetic receptor comprising the actual ligand-binding pocket of SR-AI. A selective peptide antagonist for SR-AI was identified, which constitutes a promising lead to the development of SR-AI targeted therapy and imaging of atherosclerotic lesions.

Scavenger Receptor-AI–Targeted Iron Oxide Nanoparticles for In Vivo MRI Detection of Atherosclerotic Lesions

“…12,27,28 Recently, our group successfully identified a small 16-mer peptide (PP1) with high specificity and affinity for SR-AI, a promising homing device for contrast agents. 19 Concordant with our previous study, PP1-conjugated USPIO was preferentially taken up by macrophages and VSMCs. Phagocytosis occurred in an SR-dependent manner because SR-AI inhibitor (fucoidan and dG 16 ) coincubation blunted the incremental uptake, which was not observed by a partial SR-AI inhibitor (dG 3 T 13 ).…”

“…19 To address its potential for human plaque imaging, we generated LDLr −/− chimeras with hematopoietic human SR-AI expression by bone marrow transplantation (>90% bone marrow reconstitution after 6 weeks). Chimeras with hematopoietic SR-AI deficiency (SR-AI −/− ) and wild-type bone marrowtransplanted LDLr −/− mice served as control.…”

“…Furthermore, intravenous injected PP1-coupled fluorescent nanocrystals were successfully accumulated in advanced atherosclerotic plaques of apolipoprotein E-deficient (apoE −/− ) mice. 19 In the current study, we report development and validation of SR-AI-targeted USPIO (T-USPIO) functionalized with the aforementioned SR-AI-specific peptide. The T-USPIO was validated in vitro on different mouse and human cell types found in atherosclerotic lesions and also in vivo in an established mouse model for atherosclerosis, as well as in a humanized model of atherosclerosis.…”

“…9 Here, we sought to investigate whether USPIO uptake by (plaque associated) macrophages could be augmented by conjugation to a newly devised peptide ligand for SR-AI ( Figure 1A), 19 a receptor previously shown to be highly expressed by this leukocyte subset in inflammatory foci within the plaque. 20 As a measure of USPIO uptake by RAW264.7 cells (300 μg Fe/mL), cell-associated iron was assessed using quantitative atomic absorption spectroscopy analysis.…”

Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival

“…In addition to activated macrophages, folate binds to receptors on normal epithelial cells and tumor cells (13). Segers et al (14) reported a peptide that binds the scavenger receptor-A on macrophages found within atherosclerotic plaques, but scavenger receptor-A is also expressed on all dendritic cells.…”