Everolimus-eluting stents stabilize plaque inflammation in vivo: assessment by intravascular fluorescence molecular imaging

Abstract

Aims: Inflammation drives atherosclerosis complications and is a promising therapeutic target for plaque stabilization. At present, it is unknown whether local stenting approaches can stabilize plaque inflammation in vivo. Here, we investigate whether everolimus-eluting stents (EES) can locally suppress plaque inflammatory protease activity in vivo using intravascular near-infrared fluorescence (NIRF) molecular imaging.

Methods and results: Balloon-injured, hyperlipidaemic rabbits with atherosclerosis received non-overlapping EES and bare metal stents (BMS) placement into the infrarenal aorta (n = 7 EES, n = 7 BMS, 3.5 mm diameter x 12 mm length). Four weeks later, rabbits received an injection of the cysteine protease-activatable NIRF imaging agent Prosense VM110. Twenty-four hours later, co-registered intravascular 2D NIRF, X-ray angiography and intravascular ultrasound imaging were performed. In vivo EES-stented plaques contained substantially reduced NIRF inflammatory protease activity compared with untreated plaques and BMS-stented plaques (P = 0.006). Ex vivo macroscopic NIRF imaging of plaque protease activity corroborated the in vivo results (P = 0.003). Histopathology analyses revealed that EES-treated plaques showed reduced neointimal and medial arterial macrophage and cathepsin B expression compared with unstented and BMS-treated plaques.

Conclusions: EES-stenting stabilizes plaque inflammation as assessed by translational intravascular NIRF molecular imaging in vivo. These data further support that EES may provide a local approach for stabilizing inflamed plaques.

Keywords: atherosclerosis; everolimus; fluorescence imaging; inflammation; molecular imaging; stent.

Figure 1

Schema of the experimental protocol assessing the effects of EES and BMS on inflammatory atherosclerosis in the aorta of New Zealand white rabbits. IVUS, intravascular ultrasound; BMS, bare metal stent; EES, everolimus-eluting stent; NIRF, near-infrared fluorescence.

Figure 2

Multimodal in vivo and ex vivo imaging of inflammatory protease activity in BMS-, EES-treated, and unstented plaque zones in the abdominal aorta of a representative rabbit. (A) X-ray angiogram of the abdominal aorta. Straight lines show the position of the BMS and the EES. Areas of IVUS–visible plaque (P1 and P2 zones) are highlighted. The blue arrow designates direction of blood flow. (B) In vivo NIRF catheter pullback showing NIRF signal intensity in arbitrary fluorescence units. The y-axis represents the angular dimension (0°–360°). The x-axis represents the longitudinal/axial dimension in millimetres. The asterisk denotes a guidewire artefact. (C) 1D angle-averaged mean NIRF signal along the longitudinal axis. (D) Fusion of the aligned longitudinal IVUS and intravascular NIRF images. (E) Ex vivo FRI at 800 nm of the resected aorta. AU, arbitrary units; Scale bar, 10 mm.

Figure 3

Ex vivo FRI analyses of plaque inflammatory cysteine protease activity in BMS-, EES-treated, and unstented plaque zones. (A–C) Ex vivo FRI alignments from three representative animals. All NIRF images were obtained with a one second exposure. Image windows optimized for individual images.

Figure 4

Quantification of the differences between (A) in vivo and (B) ex vivo NIRF inflammation signals among unstented plaque, BMS- and EES-treated atheroma (n = 7 rabbits). TBR, target-to-background. *P < 0.05.

Figure 5

Histological assessment of macrophages and morphometry differences among resin-embedded plaque, BMS and EES segments. Macrophages were detected by RAM-11 immunohistochemical staining (n = 37 sections analysed). (A) Total macrophage area (neointima plus media, mm²). (B) Neointimal macrophage area (mm²). (C) Medial macrophage area (mm²). (D) Neointimal area in BMS and EES (mm²). (E) Luminal stenosis (%) in BMS and EES. *P < 0.05.

Figure 6

Immunohistochemical detection of cathepsin B and macrophages from cryosections of plaque, BMS and EES segments. (A) Low magnification (2×) images of cryosections from a plaque demonstrating abundant immunoreactive macrophage RAM-11 and cathepsin B. (B, C) Immunohistochemical macrophage and cathepsin B expression, and morphological image, of a (B) BMS-treated segment and (C) EES-treated segment. Inset magnification, ×20.