Molecular imaging of atherosclerotic plaque with (64)Cu-labeled natriuretic peptide and PET

Abstract

Cardiovascular disease is the leading cause of death worldwide. PET has the potential to provide information on the biology and metabolism of atherosclerotic plaques. Natriuretic peptides (NPs) have potent antiproliferative and antimigratory effects on vascular smooth-muscle cells (VSMCs) and, in atherosclerosis, participate in vascular remodeling, in which the expression of NP clearance receptors (NPR-Cs) is upregulated both in endothelium and in VSMCs.

Methods: We investigated the potential of a C-type atrial natriuretic factor (C-ANF) to image developing plaque-like lesions in vivo. C-ANF was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and labeled with (64)Cu for noninvasive PET in a hypercholesterolemic rabbit with atherosclerotic-like lesions induced by air desiccation of a femoral artery, followed by balloon overstretch of the developing neointima. Histopathology and immunohistochemistry were performed to assess plaque development and NPR-C localization.

Results: (64)Cu-DOTA-C-ANF uptake in the atherosclerotic region was visible on small-animal PET images, with the highest target-to-background ratio (3.59 +/- 0.94) observed after the air desiccation-induced injury. Immunohistochemistry and immunofluorescence staining showed NPR-C near the luminal surface of the plaque and in VSMCs. PET and immunohistochemistry competitive blocking studies confirmed receptor-mediated tracer uptake in the plaque. With blocking, PET tracer localization of atherosclerotic to control arteries was decreased from 1.42 +/- 0.02 to 1.06 +/- 0.06 (P < 0.001).

Conclusion: We demonstrated that (64)Cu-DOTA-C-ANF is a promising candidate tracer for in vivo PET of NPR-Cs on atherosclerotic plaques.

Figure 1

Schematic diagram of experimental design.

Figure 2

Blood clearance of ⁶⁴Cu-DOTA-C-ANF in rabbits.

Figure 3

Light micrographs of femoral arterial cross-sections from hypercholesterolemic rabbits obtained at 3 TPs after injury and stained with Verhoeff's Van Gieson for elastin and fluorescent images of corresponding sections immunostained for NPR-C or blocked before immunostaining. Low-power micrographs are at ×4 magnification, high-power insets are ×400 magnification. L = lumen. (A) Control femoral artery from TP 2 rabbit shown in C. Inset shows intact internal elastic lamina (IEL), medium (M), and adventitia (A). Uninjured, control femoral artery shows only IEL autofluorescence. Control femoral artery immunofluorescence after competitive blocking of antibody–antigen binding shows no difference, compared with preblocked image. (B) Injured femoral artery at TP 1. Inset shows primary neointima (1° NEO) containing numerous foam cells (FCs) and dark nuclei of VSMC. Immunohistochemistry shows increased fluorescence near luminal or endothelial surface of primary neointima (arrow). TP 1 immunofluorescence after competitive blocking of antibody–antigen binding indicates specific binding of primary antibody to NPR-C. (C) Injured femoral artery at TP 2 showing broken IEL (adjacent to inset box) and development of amorphous, less cellular secondary neointima (2° NEO), shown in inset. Endothelium is artifactually lifted away from neointima. Immunohistochemistry shows some fluorescence on less cellular secondary neointima. (D) Injured femoral artery at TP 3 showing enlarged 2° NEO comprising predominantly matrix and elastin elements, with few cells. Immunohistochemistry shows some fluorescence on less cellular secondary neointima. In all immunostained images, IEL demonstrates autofluorescence. IHC = immunohistochemistry.

Figure 4

Micrographs of NPR-C colocalization with VSMC and endothelium at TP 1 and TP 2. (A) NPR-C (green) colocalized with VSMC (red) using anti–α-smooth muscle actin (SM) at TP 1. (B) NPR-C (green) colocalized with endothelium (red) using VCAM-1. (C) NPR-C colocalized with VSMC using SM at TP 2. (D) NPR-C colocalized with endothelium (red) using VCAM-1 at TP 2. N = neointimal proliferation. *VSMC–neointimal proliferation interface.

Figure 5

MR T1-weighted transverse image of typical atherosclerotic lesion in femoral artery of rabbit at TP 1.

Figure 6

Uptake of ⁶⁴Cu-DOTA-C-ANF on injured (yellow arrow and curve) and control (green arrow and curve) arteries in rabbits from TP 1. (A) Representative transverse PET slice, preblocking study. (B) Representative transverse PET slice, blocking study. (C) Time–activity curves of image in A. (D) Time–activity curves of image in B. F (arrow) = fiducial marker.

Figure 7

(A) ⁶⁴Cu-DOTA-C-ANF tracer uptake SUV on injured femoral arteries, noninjured control arteries, and surrounding muscle at 3 experimental TPs, representing progression and remodeling of atherosclerotic plaques. (B) SUV ratios of tracer uptake at each TP (TP 1, n = 9; TP 2, n = 6; and TP 3, n = 4).