Characterization of VCAM-1-Binding Peptide-Functionalized Quantum Dots for Molecular Imaging of Inflamed Endothelium

Yun Chen,Peter Friberg,Mátyás Molnár,Li Li,Li-Ming Gan,Ying Fu,Hjalmar Brismar,David S Milstone

doi:10.1371/journal.pone.0083805

Abstract

Inflammation-induced activation of endothelium constitutes one of the earliest changes during atherogenesis. New imaging techniques that allow detecting activated endothelial cells can improve the identification of persons at high cardiovascular risk in early stages. Quantum dots (QDs) have attractive optical properties such as bright fluorescence and high photostability, and have been increasingly studied and developed for bio-imaging and bio-targeting applications. We report here the development of vascular cell adhesion molecule-1 binding peptide (VCAM-1 binding peptide) functionalized QDs (VQDs) from amino QDs. It was found that the QD fluorescence signal in tumor necrosis factor (TNF-) treated endothelial cells in vitro was significantly higher when these cells were labeled with VQDs than amino QDs. The VQD labeling of TNF--treated endothelial cells was VCAM-1 specific since pre-incubation with recombinant VCAM-1 blocked cells' uptake of VQDs. Our ex vivo and in vivo experiments showed that in the inflamed endothelium, QD fluorescence signal from VQDs was also much stronger than that of amino QDs. Moreover, we observed that the QD fluorescence peak was significantly blue-shifted after VQDs interacted with aortic endothelial cells in vivo and in vitro. A similar blue-shift was observed after VQDs were incubated with recombinant VCAM-1 in tube. We anticipate that the specific interaction between VQDs and VCAM-1 and the blue-shift of the QD fluorescence peak can be very useful for VCAM-1 detection in vivo.

Highlights

Atherosclerosis develops over decades and is often silent until an acute event occurs in later life
In our early studies where we used un-functionalized Quantum dots (QDs) to label rat endothelial progenitor cells (EPCs) [22], we found a blueshift in the fluorescence peak of QDs located inside EPCs as compared with QDs located outside EPCs [23]
We further evaluated the VQDs in a mouse model with an increased endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) induced by lipopolysaccharide (LPS) [28]

Summary

Introduction

Atherosclerosis develops over decades and is often silent until an acute event occurs in later life. In the early stages of atherosclerosis, the endothelial layer lining the lumen of the vessel undergoes a series of changes that are activated by inflammation, e.g., the increased expression of adhesion molecules at the endothelial cell surface [1,2] Adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), play an important role in the rolling and tethering of leukocytes on the vessel wall and the subsequent accumulation of macrophage-derived foam cells and the formation of atherosclerotic lesion in the vessel wall [3]. This provides a basis for a new approach to develop imaging agents that can be specific for abnormal endothelial cells, offering excellent opportunities for molecular imaging of early atherosclerosis. Fluorophores and magnetic particles functionalized with VCAM-1 binding peptide or anti-VCAM-1 antibody have already been used to visualize the VCAM-1 expressing endothelium [4,5,6]

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Discussion

Conclusion