Two-dimensional intravascular near-infrared fluorescence molecular imaging of inflammation in atherosclerosis and stent-induced vascular injury

Abstract

Objectives: This study sought to develop a 2-dimensional (2D) intravascular near-infrared fluorescence (NIRF) imaging strategy for investigation of arterial inflammation in coronary-sized vessels.

Background: Molecular imaging of arterial inflammation could provide new insights into the pathogenesis of acute myocardial infarction stemming from coronary atheromata and implanted stents. Presently, few high-resolution approaches can image inflammation in coronary-sized arteries in vivo.

Methods: A new 2.9-F rotational, automated pullback 2D imaging catheter was engineered and optimized for 360° viewing intravascular NIRF imaging. In conjunction with the cysteine protease-activatable imaging reporter Prosense VM110 (VisEn Medical, Woburn, Massachusetts), intra-arterial 2D NIRF imaging was performed in rabbit aortas with atherosclerosis (n =10) or implanted coronary bare-metal stents (n = 10, 3.5-mm diameter, day 7 post-implantation). Intravascular ultrasound provided coregistered anatomical images of arteries. After sacrifice, specimens underwent ex vivo NIRF imaging, fluorescence microscopy, and histological and immunohistochemical analyses.

Results: Imaging of coronary artery-scaled phantoms demonstrated 8-sector angular resolution and submillimeter axial resolution, nanomolar sensitivity to NIR fluorochromes, and modest NIRF light attenuation through blood. High-resolution NIRF images of vessel wall inflammation with signal-to-noise ratios >10 were obtained in real-time through blood, without flushing or occlusion. In atherosclerosis, 2D NIRF, intravascular ultrasound-NIRF fusion, microscopy, and immunoblotting studies provided insight into the spatial distribution of plaque protease activity. In stent-implanted vessels, real-time imaging illuminated an edge-based pattern of stent-induced arterial inflammation.

Conclusions: A new 2D intravascular NIRF imaging strategy provides high-resolution in vivo spatial mapping of arterial inflammation in coronary-sized arteries and reveals increased inflammation-regulated cysteine protease activity in atheromata and stent-induced arterial injury.

Figure 1

Schematic of the constructed 2D NIRF imaging system. The tip of the fiber contains a right angle prism that reflects the guides laser light into the artery wall, and couples the subsequent fluorescent light back into the fiber. The fluorescent light is then directed to a dichroic beam-splitter that selectively reflects it into a photomultiplier tube. The beam passes additional filters to minimize the parasitic signals of laser photons and auto-fluorescence. The inset shows the spectra of the three filters (I,II,III) used in the system.

Figure 2

Evaluation of the 2D NIRF apparatus in coronary artery scale phantoms. The SNR of the catheter for different fluorochrome concentrations measured in (A) saline and (B) blood-like solutions. (C) Angular and (D) axial resolution of the system in saline and blood-like solutions. The dashed line in A and B designates the sensitivity limit of the system, corresponding to SNR=4. d, distance from catheter-to-target; the fiber-to-target distance equals d+0.5mm.

Figure 3

In vivo high-resolution NIRF molecular imaging of cysteine proteinase activity in atherosclerosis. (A) Aorta from atherosclerotic rabbits injected with Prosense VM110 one day prior, and imaged in vivo with the intravascular NIRF catheter. (B) Ex vivo FRI at 800 nm and (C) ex vivo NIRF-white light fusion image. (D) Aortas from atherosclerotic rabbits injected with saline and imaged in vivo with the NIRF catheter, and (E) ex vivo FRI at 800nm and (F) ex vivo NIRF-white light fusion image. (G) Normal rabbit injected with VM110 and imaged with the intravascular NIRF catheter. (H) Ex vivo FRI at 800nm (1 sec) and (I) ex vivo NIRF-white light fusion image. In vivo and ex vivo fluorescence images were equally windowed and processed (color lookup table applies to all figures in each row). Scale bar, 10 mm.

Figure 4

Ex vivo NIR fluorescence reflectance imaging analyses of atheroma inflammation. The three experimental groups tested were: atherosclerosis+Prosense VM110 (Athero/VM110), atherosclerosis+saline (Athero+saline), and normal+Prosense VM110 (Normal/VM110), where Prosense VM110 is a protease-activatable NIRF imaging agent. (A) Ex vivo vessel SNR, (B) plaque TBR, and (C) plaque SNR from the three experimental groups. Athero=atherosclerosis. *p<0.05.

Figure 5

Representative in vivo molecular and anatomical imaging of inflamed atheromata. (A) Angiographic appearance (with inset high magnification image in dotted yellow lines) of radiopaque tip of NIRF catheter positioned just proximal to the iliac bifurcation, enabling co-registration with IVUS. (B) Angiogram of the atherosclerotic iliac and aorta. Tip of the NIRF catheter at pullback initiation confirming its intravascular position (colored yellow). (C) Longitudinal IVUS image of the abdominal aortoiliac arteries. Arrows demarcate IVUS-detectable mildly stenotic plaques (P₁,P₂). (D) Image of NIRF catheter pullback aligned with the angiogram and IVUS image demonstrates NIRF signal in small volume plaques, in >3.0 mm diameter arteries. Corresponding 1D plot of the angle-averaged 2D NIRF intensity pullback below. (E) Longitudinal superimposed NIRF and IVUS fusion images (yellow/white=strongest NIRF signal intensity, red/black=lowest NIRF signal intensity). (F,G) High magnification image of plaque zones P₁ and P₂. Arrows indicate minimally stenotic atherosclerotic plaques. (H, I) Axial IVUS images from zone P₁ and P₂.

Figure 6

NIRF signal analyses of atheroma inflammation detected in vivo by the 2D NIRF catheter. (A,B,C) In vivo plaque TBR, SNR and vessel SNRs from atherosclerotic animals injected with either Prosense VM110 (Athero+VM110) or saline (Athero+saline), or from normal (non-atherosclerotic) animals injected with Prosense VM110 (normal+VM110). *p<0.05.

Figure 7

Histopathological and immunoblotting assessments of aortic inflammation. In each row, the five images depict correlative arterial sections of hematoxylin and eosin staining (10X), immunoreactive macrophages (RAM-11, 20X), immunoreactive cathepsin B (catB, 20X), NIR fluorescence at 800nm, and merged 800nm-535 nm fluorescence, with red color denoting 800nm signal induced by cysteine protease activation of Prosense VM110, and blue color denoting signal at 535nm originating from autofluorescence. (A) In an advanced plaque, immunoreactive macrophages and cathepsin B, and intimal NIRF protease activity signals are evident. (B) Early stage atheroma demonstrates macrophages and cathepsin B in the intima and media but little NIRF cysteine protease activity (800nm image). (C) Section of a normal artery. Elastin fibers provide a source of autofluorescence (blue color) on fusion fluorescence microscopy images. (D) Cathepsin B and β-actin immunoblot of lysates from normal and plaque containing vessel demonstrates cathepsin B in atherosclerotic aortas. Pro-catB denotes the 46 kD pre-cathepsin B band, and mature catB denotes the 25 kD and 30 kD cathepsin B bands.

Figure 7

Histopathological and immunoblotting assessments of aortic inflammation. In each row, the five images depict correlative arterial sections of hematoxylin and eosin staining (10X), immunoreactive macrophages (RAM-11, 20X), immunoreactive cathepsin B (catB, 20X), NIR fluorescence at 800nm, and merged 800nm-535 nm fluorescence, with red color denoting 800nm signal induced by cysteine protease activation of Prosense VM110, and blue color denoting signal at 535nm originating from autofluorescence. (A) In an advanced plaque, immunoreactive macrophages and cathepsin B, and intimal NIRF protease activity signals are evident. (B) Early stage atheroma demonstrates macrophages and cathepsin B in the intima and media but little NIRF cysteine protease activity (800nm image). (C) Section of a normal artery. Elastin fibers provide a source of autofluorescence (blue color) on fusion fluorescence microscopy images. (D) Cathepsin B and β-actin immunoblot of lysates from normal and plaque containing vessel demonstrates cathepsin B in atherosclerotic aortas. Pro-catB denotes the 46 kD pre-cathepsin B band, and mature catB denotes the 25 kD and 30 kD cathepsin B bands.

Figure 8

Representative multimodality NIRF molecular and IVUS anatomical imaging of arterial inflammation at day 7 following coronary stent implantation. (A) Angiogram of an implanted bare metal stent in the abdominal aorta. Dotted rectangle denotes stent position. (B,C) Longitudinal IVUS and NIRF catheter pullbacks demonstrate NIRF signal within the stent. NIRF signal collection was performed through blood without flushing, in 3.5mm diameter vessels. (D) Corresponding 1D plot of the angle-averaged 2D NIRF signal at each axial point. (E) Longitudinal IVUS and NIRF fusion image (yellow/white=strongest NIRF signal intensity, red/black=lowest NIRF signal intensity). (F,G,H) Axial IVUS images from PTCA alone, stent and normal vessel zones, respectively.

Figure 8

Representative multimodality NIRF molecular and IVUS anatomical imaging of arterial inflammation at day 7 following coronary stent implantation. (A) Angiogram of an implanted bare metal stent in the abdominal aorta. Dotted rectangle denotes stent position. (B,C) Longitudinal IVUS and NIRF catheter pullbacks demonstrate NIRF signal within the stent. NIRF signal collection was performed through blood without flushing, in 3.5mm diameter vessels. (D) Corresponding 1D plot of the angle-averaged 2D NIRF signal at each axial point. (E) Longitudinal IVUS and NIRF fusion image (yellow/white=strongest NIRF signal intensity, red/black=lowest NIRF signal intensity). (F,G,H) Axial IVUS images from PTCA alone, stent and normal vessel zones, respectively.

Figure 9

NIRF imaging of stent injury. (A) Ex vivo FRI at 800nm reveals augmented NIRF protease activity at stent edges. (B) Corresponding ex vivo intravascular NIRF pullback also detected stent-based NIRF signal increases. (C) Ex vivo NIRF-white light fusion image of the stent, (D) high-magnification white light and (E) high-magnification NIRF image reveals signal along the greater curvature of stent struts. (F) In vivo and (G) ex vivo SNR in the normal and stented aorta (paired observations). (H) Immunoblot of cathepsin B (catB) staining in normal and stented rabbit aorta. Pro-catB denotes the 46 kD pre-cathepsin B band, and mature catB denotes the 25 kDa and 30 kDa cathepsin B bands. Scale bar, 10 mm. *p<0.05.