Molecular imaging of atherosclerotic plaques targeted to oxidized LDL receptor LOX-1 by SPECT/CT and magnetic resonance

Abstract

Background: The oxidized low-density lipoprotein receptor (LDLR) LOX-1 plays a crucial role in atherosclerosis. We sought to detect and assess atherosclerotic plaque in vivo by using single-photon emission computed tomography/computed tomography and magnetic resonance imaging and a molecular probe targeted at LOX-1.

Methods and results: Apolipoprotein E(-/-) mice fed a Western diet and LDLR(-/-) and LDLR(-/-)/LOX-1(-/-) mice fed an atherogenic diet were used. Imaging probes consisted of liposomes decorated with anti-LOX-1 antibodies or nonspecific immunoglobulin G, (111)indium or gadolinium, and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine fluorescence markers. In vivo imaging was performed 24 hours after intravenous injection (150 microL) of LOX-1 or nonspecific immunoglobulin G probes labeled with either (111)indium (600 muCi) or gadolinium (0.075 mmol/kg), followed by aortic excision for phosphor imaging and Sudan IV staining, or fluorescence imaging and hematoxylin/eosin staining. The LOX-1 probe also colocalized with specific cell types, apoptosis, and matrix metalloproteinase-9 expression in frozen aortic sections. Single-photon emission computed tomography/computed tomography imaging of the LOX-1 probe showed aortic arch "hot spots" in apolipoprotein E(-/-) mice (n=8), confirmed by phosphor imaging. Magnetic resonance imaging showed significant Gd enhancement in atherosclerotic plaques in LDLR(-/-) mice with the LOX-1 (n=7) but not with the nonspecific immunoglobulin G (n=5) probe. No signal enhancement was observed in LDLR(-/-)/LOX-1(-/-) mice injected with the LOX-1 probe (n=5). These results were confirmed by ex vivo fluorescence imaging. The LOX-1 probe bound preferentially to the plaque shoulder, a region with vulnerable plaque features, including extensive LOX-1 expression, macrophage accumulation, apoptosis, and matrix metalloproteinase-9 expression.

Conclusions: LOX-1 can be used as a target for molecular imaging of atherosclerotic plaque in vivo. Furthermore, the LOX-1 imaging signal is associated with markers of rupture-prone atherosclerotic plaque.

Figure 1

LOX-1 probe (¹¹¹In-liposome-LOX-1 Antibody-DiI) binding to LOX-1 antigen in vitro. Left top panel is a representative dot blot analysis. The mean data from 3 separate experiments are shown in the left lower panel. Right panel shows that LOX-1 probe (Gd-liposome-LOX-1 antibody-DiI) bound to LOX-1 antigen in vitro by ELISA. The non-specific IgG (nIgG) probe (Gd-liposome-nIgG-DiI) showed no binding to LOX-1 antigen (n=3).

Figure 2

Blood pool clearance of probes in C57BL6 and Apo E^−/− mice (top panel). Blood pool clearance of LOX-1 and nIgG probes was similar in wild type C57BL6 mice (left upper panel). LOX-1 probes cleared slowly compared to nIgG probes in the Apo E^−/− mice. However, there was no difference in clearance between the two probes at 24 hrs (right upper panel). Bottom panel shows tissue bio-distribution. LOX-1 probes accumulated mainly in liver and spleen.

Figure 3

LOX-1 expression and LOX-1 probe binding in atherosclerotic plaque. LOX-1 is highly expressed in atherosclerotic plaques, especially in the shoulder region (left panel). The LOX-1 probe bound mainly to the shoulder area of the atherosclerotic plaque (middle panel). There was little binding of the nIgG probe (right panel). L – lumen.

Figure 4

Confocal microscopic images of aortic sections. LOX-1 probe in plaque appears red; macrophages appear green; smooth muscle cells appear purple and nuclei are blue. LOX-1 probe is co-localized mainly with macrophages in the atherosclerotic plaque (yellow = overlap of red and green). There was little LOX-1 probe co-localization with proliferating smooth muscle cells.

Figure 5

LOX-1, apoptosis and MMP9 expression in the plaque. LOX-1 probe binding (yellow color) was co-localized with apoptotic cells (green, left panel) and with cells expressing MMP9 (green, right panel).

Figure 6

SPECT(A) and ex vivo phosphor imaging (C) showed no focal aortic arch hotspots in Apo E^−/− mice injected with nIgG probe, whereas all Apo E^−/− mice injected with LOX-1 probe had hotspots in the aortic arch (B- sagittal, coronal and transverse planes), confirmed by ex vivo phosphor imaging (D). Sudan IV staining demonstrated comparable plaques between the two groups (E&F).

Figure 7

MRI showed post-injection gadolinium enhancement in aortic root to arch in the LDLR^−/− mice injected with LOX-1 (D), but not nIgG probes (E), compared to pre-injection imaging (A & B). Fluorescence imaging of frozen sections confirmed LOX-1 probe binding (yellow color) in the same plaque area (G) with little nonspecific nIgG probe binding (H). Post-gadolinium enhancement was not seen in LDLR ^−/−/LOX-1^−/− double knockout mice injected with LOX-1 probes (F vs C), nor was LOX-1 probe binding seen in plaque from these mice (I). H&E staining (bottom row) shows the size of atherosclerotic plaque from adjacent fluorescent imaging sections. Quantitative MRI data (right panel) showed that LDLR^−/− mice injected with LOX-1 probe increased contrast to noise ratio (CNR) and normalized enhancement ratio (NER) compared with the two control groups. PA-pulmonary artery. AA- ascending aorta.