Molecular imaging with optical coherence tomography using ligand-conjugated microparticles that detect activated endothelial cells: rational design through target quantification

Abstract

Objectives: Optical coherence tomography (OCT) is a high resolution imaging technique used to assess superficial atherosclerotic plaque morphology. Utility of OCT may be enhanced by contrast agents targeting molecular mediators of inflammation.

Methods and results: Microparticles of iron oxide (MPIO; 1 and 4.5 μm diameter) in suspension were visualized and accurately quantified using a clinical optical coherence tomography system. Bound to PECAM-1 on a plane of cultured endothelial cells under static conditions, 1 μm MPIO were also readily detected by OCT. To design a molecular contrast probe that would bind activated endothelium under conditions of shear stress, we quantified the expression (basal vs. TNF-activated; molecules μm(-2)) of VCAM-1 (not detected vs. 16 ± 1); PECAM-1 (132 ± 6 vs. 198 ± 10) and E-selectin (not detected vs. 46 ± 0.6) using quantitative flow cytometry. We then compared the retention of antibody-conjugated MPIO targeting each of these molecules plus a combined VCAM-1 and E-selectin (E+V) probe across a range of physiologically relevant shear stresses. E+V MPIO were consistently retained with highest efficiency (P < 0.001) and at a density that provided conspicuous contrast effects on OCT pullback.

Conclusion: Microparticles of iron oxide were detectable using a clinical OCT system. Assessment of binding under flow conditions recommended an approach that targeted both E-selectin and VCAM-1. Bound to HUVEC under conditions of flow, targeted 1 μm E+V MPIO were readily identified on OCT pullback. Molecular imaging with OCT may be feasible in vivo using antibody targeted MPIO.

Supplementary Fig. 1

Upregulation of E-selectin and VCAM-1 in TNF-α stimulated HUVEC, and constitutive expression of PECAM-1. Immunofluorescence microscopy demonstrating presence and localisation of E-selectin and VCAM-1 in TNF-α stimulated cells and PECAM-1 in basal cells (A–C respectively). Real-time RT-PCR of basal (−) and TNF-α stimulated (10 ng mL⁻¹ of TNF-α for 4 h) (+) HUVEC and HUVEC-C confirms inducible expression of E-selectin and VCAM-1 in stimulated but not basal HUVEC and constitutive expression of PECAM-1 in both basal and stimulated cells (D–F).

Supplementary Fig. 2

Quantitative flow cytometry to establish relative ligand density on HUVEC. Histograms generated from counts of fluorescent labelling of cells only (negative control), CD68 (isotype control), VCAM-1, E-selectin and PECAM-1 on the surface of basal (top row) and TNF-α stimulated (bottom row) HUVEC. Fold-difference of fluorescence intensity relative to the cells only control was calculated by the Cytobank web-based analysis software and a heatmap generated. The relative ligand densities were calculated from linear regression analysis of the Qifikit calibration beads and displayed in Supplementary Table 1.

Supplementary Fig. 3

Antibody-labelling and flow cytometry evaluation of MPIO conjugation. The antibody labelling of MPIO was verified by labelling with an Alexa 488-conjugated anti-mouse secondary antibody and images taken with a monochrome CCD camera. MPIO labelled with antibodies against E-selectin, VCAM-1, a combination of E-selectin and VCAM-1, PECAM-1 and a non-specific IgG₂ control were imaged separately and pseudocoloured red, green, yellow, magenta and cyan, respectively (A). Quantitative flow cytometry was used to evaluate loading efficiency. For all antibodies labelled, approximately 5–10% remained unlabelled, while the labelled MPIO comprised single MPIO and multiples, resulting in multiple peaks (B) (example shown is E-selectin–MPIO). Differential interference contrast images of antibody-labelled MPIO confirm a mixture of single and multiple particles (C).

Fig. 1

Contrast optical coherence tomography. The commercially available LightLab C7-XR OCT Intravascular Imaging System used in this study (A). Optical coherence tomography reveals minimal background scatter (B) of the 0.4% agarose used in the preparation of MPIO phantoms. 4.5 μm MPIO at variable concentration in an arterial phantom (C) were quantifiable using automated analysis of OCT data (D), and visible as punctuate spots in the image (E). 1 μm MPIO in a phantom (F) were also quantifiable (G), and visible as punctuate spots in the image (H).

Fig. 2

Optical coherence tomography of MPIO on a cell monolayer. (A) A cartoon representation of antibody-labelled MPIO [red] binding to endothelial cell surface markers [yellow]. The PECAM-1–MPIO bound to the cell monolayer are visible as a bright horizontal band in the OCT image (B). ‘No-MPIO’ control experiment confirms background signal is minimal (C). The intensity profile of a line drawn perpendicular to the monolayer with PECAM-1–MPIO bound reveals a clear demarcation between MPIO and surrounding agarose and cells (D). Areas of measurable signal along the pullback are significantly greater for PECAM-1–MPIO compared with the no MPIO control (E) (Error bars represent ±1 SEM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Binding of E-selectin-, VCAM-1-, E + V-, PECAM-1- and IgG₂–MPIO to TNF-α stimulated and basal HUVEC. In stimulated cells under static and low shear stress conditions, antibody–MPIO to E-selectin, VCAM-1 and E + V bind in greater numbers than PECAM-1–MPIO. However, as shear stress increases, PECAM-1–MPIO bind in significantly greater numbers than the other antibody–MPIO (A). In basal cells, only antibody–MPIO targeted to the constitutively expressed PECAM-1 are able to bind above background (B). Data points are expressed as mean MPIO bound per field of view (MPIO/fov), with error bars representing ±1 SEM.

Fig. 4

Representative microphotographs show isolated, pressurized coronary arterioles (internal diameter: 100 μm) of the rat. Left panel shows bright field image of the arteriole, whereas fluorescent (FITC excited) images show VCAM-1–MPIO (arrowheads) adhered to the endothelial cells before (basal, shown in the middle panel) and after stimulation with TNF-α (7.25 nmol L⁻¹ for 4-h, right panel). VCAM-1–MPIO were delivered intraluminally into the pressurized arteriole at the flow rate of 25 μL min⁻¹ (A). Mean binding events in isolated coronary arterioles were significantly higher in TNF-α stimulated vessel than in basal tissue (B). Western immunoblots show expression of VCAM-1 and β-actin in coronary arterioles before (basal) and after stimulation with TNF-α (7.25 nmol L⁻¹ for 4-h).

Fig. 5

OCT imaging of MPIO binding to E-selectin and VCAM-1 under conditions of shear stress. Binding of E + V–MPIO at 1 dyne cm⁻² was detectable using OCT, and the particles bound to the cell monolayer were clearly visible (A). No signal was seen in the basal control at 1 dyne cm⁻² (B). The intensity profile of a line drawn perpendicular to the monolayer with PECAM-1–MPIO bound reveals a clear demarcation between MPIO and surrounding agarose and cells (C). Area of signal generated by the E + V–MPIO binding at 1 dyne cm⁻² in the OCT images is significantly higher in the stimulated cells than in the basal controls (D&E), while the reduction in binding to stimulated cells at 5 dyne cm⁻² results in a lower signal area, but which is still significantly greater than the basal equivalent (E). Error bars represent ±1 SEM.