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. 2008 Jan;28(1):77-83.
doi: 10.1161/ATVBAHA.107.145466. Epub 2007 Oct 25.

Magnetic resonance imaging of endothelial adhesion molecules in mouse atherosclerosis using dual-targeted microparticles of iron oxide

Martina A McAteer  1 , Jurgen E Schneider, Ziad A Ali, Nicholas Warrick, Christina A Bursill, Constantin von zur Muhlen, David R Greaves, Stefan Neubauer, Keith M Channon, Robin P Choudhury
Affiliations

Magnetic resonance imaging of endothelial adhesion molecules in mouse atherosclerosis using dual-targeted microparticles of iron oxide

Martina A McAteer et al. Arterioscler Thromb Vasc Biol. 2008 Jan.

Abstract

Objective: Microparticles of iron oxide (MPIO) distort magnetic field creating marked contrast effects far exceeding their physical size. We hypothesized that antibody-conjugated MPIO would enable magnetic resonance imaging (MRI) of endothelial cell adhesion molecules in mouse atherosclerosis.

Methods and results: MPIO (4.5 microm) were conjugated to monoclonal antibodies against vascular cell adhesion molecule-1 (VCAM-MPIO) or P-selectin (P-selectin-MPIO). In vitro, VCAM-MPIO bound, in dose-dependent manner, to tumor necrosis factor (TNF)-alpha stimulated sEND-1 endothelial cells, as quantified by light microscopy (R2=0.94, P=0.03) and by MRI (R2=0.98, P=0.01). VCAM-MPIO binding was blocked by preincubation with soluble VCAM-1. To mimic leukocyte binding, MPIO targeting both VCAM-1 and P-selectin were administered in apolipoprotein E-/- mice. By light microscopy, dual-targeted MPIO binding to endothelium overlying aortic root atherosclerosis was 5- to 7-fold more than P-selectin-MPIO (P<0.05) or VCAM-MPIO (P<0.01) alone. Dual-targeted MPIO, injected intravenously in vivo bound aortic root endothelium and were quantifiable by MRI ex vivo (3.5-fold increase versus control; P<0.01). MPIO were well-tolerated in vivo, with sequestration in the spleen after 24 hours.

Conclusions: Dual-ligand MPIO bound to endothelium over atherosclerosis in vivo, under flow conditions. MPIO may provide a functional MRI probe for detecting endothelial-specific markers in a range of vascular pathologies.

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Figures

Figure 1
Figure 1
Anti-VCAM-1-MPIO binding to TNF-α stimulated sEND-1 cells. (A) Light microscopy images of anti-VCAM-1-MPIO binding to TNF-α stimulated sEND-1 cells (0.1-100 ng / mL) (x 20 magnification). MPIO binding persisted after extensive washing and was restricted to cellular areas. A dose-dependent increase in anti-VCAM-1-MPIO binding in response to increasing TNF-α stimulation was observed. Data are mean ± SD of duplicate experiments (4 fields per sample). (B) High resolution MRI (11.7 T; resolution 25 × 25 × 25 μm) of MPIO-bound sEND-1 cell phantoms confirmed the dose response of anti-VCAM-1-MPIO binding to TNF-α stimulated sEND-1 cells. Data are mean ± SD for 8 MR images per sample, at intervals of 293 μm.
Figure 2
Figure 2
Competitive inhibition of anti-VCAM-1-MPIO binding to TNF-α (50 ng / mL) stimulated sEND-1 cells in vitro. (A) Pre-incubation of anti-VCAM-1-MPIO with soluble VCAM-1 protein (Fc-VCAM-1) inhibited MPIO binding almost entirely (P<0.001), while pre-incubation with Fc-ICAM-1 had no effect. Data are mean ± SD for 4 fields per sample (triplicate experiments). (B) Confocal microscopy confirmed cell surface expression of VCAM-1 (green fluorescence) in TNF-α stimulated cells. Fc-ICAM-1 had no effect on anti-VCAM-MPIO binding (autofluorescent green spheres) while Fc-VCAM-1 abolished anti-VCAM-1-MPIO retention, despite demonstrable cell surface VCAM-1 expression. Blue represents DAPI staining for cell nuclei.
Figure 3
Figure 3
High resolution ex vivo MRI (11.7 T; resolution 29 × 29 × 29 μm) of macrophage or sEND-1 cell phantoms pre-incubated with MPIO for 30 min or 2 h. Data are mean ± SD of low signal areas in 10 MR images per sample at intervals of 293 μm. Low signal areas, corresponding to MPIO uptake by cells, were detected for macrophages pre-incubated with MPIO for 30 min or 2 h but not for sEND-1 cells (***P<0.001). No significant difference was detected for sEND-1 cell phantoms pre-incubated with MPIO for 30 min or 2 h and the negative control sEND-1 ‘cell only’ phantom, not exposed to MPIO.
Figure 4
Figure 4
(A) Histological examination of aortic roots following left ventricular injection of single versus dual-targeted MPIO in terminally anaesthetised apoE-/- mice with beating hearts. (A) Three to four aortic root sections per mouse (15 μm thick) were examined for MPIO binding by light microscopy (x 40 magnification). There was minimal non-specific retention of control IgG-1-MPIO (n = 3 mice). Anti-VCAM-1-MPIO (n = 7 mice) and anti-P-selectin MPIO (n = 4 mice) showed similar levels of binding to aortic root plaque endothelium. However, dual-ligand MPIO (n = 6 mice) targeting VCAM-1 and P-selectin showed synergistically enhanced binding compared to anti-P-selectin MPIO or anti-VCAM-1-MPIO, respectively. (B) Light microscopy image depicting dense dual-targeted MPIO binding to endothelium overlying atherosclerotic plaque in the aortic root (x 40 magnification). The MPIO are clearly distinguished as bright yellow spheres confined to the luminal surface of the vessel. The dark red bodies of irregular shape within the plaque are cell nuclei. Scale bar 20 μm. (*** P < 0.001; ** P < 0.01 and * P < .05).
Figure 5
Figure 5
High resolution ex vivo MRI (11.7 T; resolution 25 × 25 × 25 μm) of aortic roots of apoE-/- mice, 30 min after i.v. injection of dual-targeted MPIO or IgG-1-MPIO (A) The number of bound dual-targeted MPIO, quantified by MRI, was 3.5-fold greater than IgG-1 MPIO (P < 0.01; n = 4 mice per group). (B) Dual-targeted MPIO were identified on MR images as distinct circular low signal areas adherent to endothelium overlying atherosclerotic plaque. A ‘halo effect’ was observed for MPIO in some images, reflecting a partial volume effect: As individual MPIO were tracked through adjacent images, a dense low signal area was always present (top panel shows 3 consecutive MR images. Red arrows indicate bound MPIO, black arrows show MPIO passing in and out of the imaging plane). Scale bar 500 μm. (C) 3-D reconstruction of segmented images. Orthogonal slices were taken horizontally through the aortic valve and vertically along the axis of the aorta (panel i). Note the bloom effect of MPIO contrast in lesion areas of the aortic root and the absence of binding in atherosclerosis-free areas of ascending aorta. In panel (ii) the vertical slice was removed to illustrate the volume of MPIO contrast in the aortic root.
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