18F-4V for PET-CT imaging of VCAM-1 expression in atherosclerosis

Abstract

Objectives: The aim of this study was to iteratively develop and validate an (18)F-labeled small vascular cell adhesion molecule (VCAM)-1 affinity ligand and demonstrate the feasibility of imaging VCAM-1 expression by positron emission tomography-computed tomography (PET-CT) in murine atherosclerotic arteries.

Background: Hybrid PET-CT imaging allows simultaneous assessment of atherosclerotic lesion morphology (CT) and may facilitate early risk assessment in individual patients. The early induction, confinement of expression to atherosclerotic lesions, and accessible position in proximity to the blood pool render the adhesion molecule VCAM-1 an attractive imaging biomarker for inflamed atheroma prone to complication.

Methods: A cyclic, a linear, and an oligomer affinity peptide, internalized into endothelial cells by VCAM-1-mediated binding, were initially derivatized with DOTA to determine their binding profiles and pharmacokinetics. The lead compound was then (18)F-labeled and tested in atherosclerotic apoE(-/-) mice receiving a high-cholesterol diet as well as wild type murine models of myocardial infarction and heart transplant rejection.

Results: The tetrameric peptide had the highest affinity and specificity for VCAM-1 (97% inhibition with soluble VCAM-1 in vitro). In vivo PET-CT imaging using (18)F-4V showed 0.31 +/- 0.02 SUV in murine atheroma (ex vivo %IDGT 5.9 +/- 1.5). (18)F-4V uptake colocalized with atherosclerotic plaques on Oil Red O staining and correlated to mRNA levels of VCAM-1 measured by quantitative reverse transcription polymerase chain reaction (R = 0.79, p = 0.03). Atherosclerotic mice receiving an atorvastatin-enriched diet had significantly lower lesional uptake (p < 0.05). Furthermore, (18)F-4V imaging in myocardial ischemia after coronary ligation and in transplanted cardiac allografts undergoing rejection showed high in vivo PET signal in inflamed myocardium and good correlation with ex vivo measurement of VCAM-1 mRNA by quantitative polymerase chain reaction.

Conclusions: (18)F-4V allows noninvasive PET-CT imaging of VCAM-1 in inflammatory atherosclerosis, has the dynamic range to quantify treatment effects, and correlates with inflammatory gene expression.

Figure 1

1A: Affinity curves for candidate ligands assessed by competition after binding to immobilized VCAM-1. CPM: Counts per minute. Mean ± 95% confidence interval. 1B: Cell uptake assay after incubation of MHEC with peptides. 1C: ApoE^−/− mice of equivalent age were injected with ¹¹¹In labeled peptides. 1D: %IDGT in aortas excised from apoE^−/− after injection of TLP, MCP, and MLP, *p<0.05.

Figure 2

2A: Three-dimensional model of ¹⁸F-4V. The tracer ¹⁸F is located on the top, and the 4 branching affinity peptides point downward. 2B: Original HPLC trace of ¹⁸F-4V.

Figure 3

3A: Pre-incubation of the probe with soluble VCAM-1 significantly reduces uptake of ¹⁸F-4V into MHEC. CPM: Counts per minute. 3B: Uptake of ¹⁸F-4V in excised aortas by scintillation counting. MAb: preinjection of a monoclonal VCAM-1–targeted antibody, *p<0.05. 3C: Exposure of aortas on a phosphorimager corroborates highest uptake of ¹⁸F-4V in apoE-deficient mice, with little uptake in wild-type aortas. Oil Red O staining shows peak uptake in plaques.

Figure 4

A fluorescent version of ¹⁸F-4V (cy5-4V) was used to explore histological distribution. The endothelial and subendothelial layers of an atherosclerotic plaque show strong uptake, while autofluorescence in the FITC channel is neglible. On adjacent sections, VCAM-1 and endothelial staining colocalize with the agent, with some uptake in macrophages and smooth muscle cells. Magnification 400×.

Figure 5

Dynamic PET imaging identified 60–120 minutes after injection as a suitable time window. Blood activity was measured in the left ventricular blood pool. Signal in the blood pool and the aortic root is plotted (A) and shown over time in short (B) and long axis (C) PET-CT.

Figure 6. PET-CT imaging in apoE^-/- and statin treated mice

A, C, E: short-axis PET-CT images of the aortic root (arrows). B, D, F: long-axis views. G, H, I: 3D maximum intensity projection (bone=white, vasculature+blue, ¹⁸F-4V=red). K: PET signal as SUV (standard uptake value), *p<0.05.

Figure 7

Correlation of ¹⁸F-4V uptake to VCAM-1 expression by quantitative RT PCR, *p<0.05.

Figure 8

8A: PET-CT 5 days after MI shows strong signal in the infarcted LV wall. 8B: Contrast-enhanced CT depicts MI (arrows). 8C: Autoradiography of myocardial ring. 8D: %IDGT in the infarct and in non-infarcted myocardium. 8E: VCAM-1 mRNA, AU: Arbitrary units. 8F, G: PET-CT of a mouse heart transplanted heterotopically into the abdominal cavity. The rejected allograft (arrow heads) shows high uptake of ¹⁸F-4V. 8H, I: Autoradiography of the graft and the orthotopic recipient heart. 8K: Uptake of ¹⁸F-4V in rejected allografts. 8L: VCAM-1 mRNA levels on day 7 after transplantation, *p<0.05.