Atheroma Susceptible to Thrombosis Exhibit Impaired Endothelial Permeability In Vivo as Assessed by Nanoparticle-Based Fluorescence Molecular Imaging

Abstract

Background: The role of local alterations in endothelial functional integrity in atherosclerosis remains incompletely understood. This study used nanoparticle-enhanced optical molecular imaging to probe in vivo mechanisms involving impaired endothelial barrier function in experimental atherothrombosis.

Methods and results: Atherosclerosis was induced in rabbits (n=31) using aortic balloon injury and high-cholesterol diet. Rabbits received ultrasmall superparamagnetic iron oxide nanoparticles (CLIO) derivatized with a near-infrared fluorophore (CyAm7) 24 hours before near-infrared fluorescence imaging. Rabbits were then either euthanized (n=9) or underwent a pharmacological triggering protocol to induce thrombosis (n=22). CLIO-CyAm7 nanoparticles accumulated in areas of atheroma (P<0.05 versus reference areas). On near-infrared fluorescence microscopy, CLIO-CyAm7 primarily deposited in the superficial intima within plaque macrophages, endothelial cells, and smooth muscle cells. Nanoparticle-positive areas further exhibited impaired endothelial barrier function as illuminated by Evans blue leakage. Deeper nanoparticle deposition occurred in areas of plaque neovascularization. In rabbits subject to pharmacological triggering, plaques that thrombosed exhibited significantly higher CLIO-CyAm7 accumulation compared with nonthrombosed plaques (P<0.05). In thrombosed plaques, nanoparticles accumulated preferentially at the plaque-thrombus interface. Intravascular 2-dimensional near-infrared fluorescence imaging detected nanoparticles in human coronary artery-sized atheroma in vivo (P<0.05 versus reference segments).

Conclusions: Plaques that exhibit impaired in vivo endothelial permeability in cell-rich areas are susceptible to subsequent thrombosis. Molecular imaging of nanoparticle deposition may help to identify biologically high-risk atheroma.

Keywords: atherosclerosis; cholesterol; endothelium; molecular imaging; thrombosis.

Figure 1. Ex vivo assessment of NIRF nanoparticle deposition in atheroma

(A) Representative ex vivo fluorescence reflectance imaging (FRI) in a rabbit 24 hours after CLIO-CyAm7 injection. White light (WL) image shows the resected aorta, stretched to premortem length determined by in vivo x-ray angiography. CLIO-CyAm7 signal evolved in atheroma (yellow bar) in contrast to minimal signal in the uninjured aorta, iliac bifurcation, or renal artery (yellow arrow). (B) Ex vivo FRI of a rabbit injected with PBS as a control exhibits minimal NIRF signal in atheroma (yellow bar). (C) Quantification of FRI signal in untriggered animals (n=6) showing significantly higher CLIO-CyAm7 signal in regions of atheroma compared to control renal artery (p=0.03). (D) Corresponding fluorescence microscopy (FM, yellow dashed line Figure 1A) of CLIO-CyAm7-injected atheroma showing luminal surface CLIO-CyAm7 signal (red) non-circumferentially distributed. (E) FM of a control atheroma (PBS injection, yellow dashed line Figure 1B) showing minimal CLIO-CyAm7 signal despite substantial atheroma detectable by autofluorescence. (F) FM analysis of percent positive CLIO-CyAm7 pixels in experimental, triggered rabbits at 48 hours showing increased CLIO-CyAm7 accumulation in regions of atheroma versus uninjured aorta without evidence of intimal thickening (p<0.0001, n=183 slices). Scale bars; FRI = 1cm, Microscopy = 1mm. CLIO-CyAm7 = red, Autofluorescence = blue.

Figure 2. Cellular distribution of CLIO-CyAm7 nanoparticles in atherosclerosis

CLIO-CyAm7 uptake by plaque endothelial cells (ECs), macrophages (Macs) and smooth muscle cells (SMCs). Carstairs’ stain shows structural characteristics of atheroma, including collagen (blue). (A) Carstairs’, immunohistochemical (IHC), and immunofluorescence (IF) stains for CD31 (A1), RAM11 (A2), and alpha-smooth muscle actin (aSMA, A3) show correspondence between CLIO-CyAm7 signal (indicated by red arrows) and superficial, luminal ECs, macrophages, and SMCs, respectively. (B) Distribution of CLIO-CyAm7 along the intimal-medial border in regions of neovasculature as detected by CD31 stain (B1). CLIO-CyAm7 deposition occurred in areas of RAM11+ macrophages flanking the neovasculature (B2). IHC: RAM11+ = red; CD31+ and aSMA+ = brown. FM fusion images: CLIO-CyAm7 = red, IF antibodies = green, autofluorescence = blue. White arrows = internal elastic membrane. Scale bars: Low magnification Carstairs’ = 100µm, IF and IHC = 25µm. L=vessel lumen.

Figure 3. Plaque endothelial permeability assessed with Evans Blue concomitantly with CLIO-CyAm7 nanoparticle deposition

A subgroup of rabbits injected with CLIO-CyAm7 also received Evans Blue shortly prior to sacrifice. (A) Atheroma with extensive Evans blue uptake (yellow) throughout the intima limited peripherally by the internal elastic membrane (autofluorescence, blue). CLIO-CyAm7 (red) similarly penetrates below the endothelial layer throughout the intima. (B) Atheroma with minimal, superficial Evans blue uptake and minimal CLIO-CyAm7 signal. (C) Evans Blue and CLIO-CyAm7 depths of penetration correlated strongly (r=0.64, p=0.003, n=19). (D) Weak correlation between the depth of Evans Blue penetration and plaque thickness (r=0.14, p=0.61, n=19). Scale bar 100µm.

Figure 4. In vivo and ex vivo NIRF imaging of atherothrombosis

Rabbits underwent the protocol described in Supplemental Figure 1. (A) Pre-trigger x-ray angiography showing the aorta for image co-registration (not shown are the iliac bifurcation and renal artery). (B) Pre-trigger in vivo NIRF imaging projected into a 2D matrix of translational distance (x-axis) and 0–360 degree of rotation (y-axis). (C,D) Pre- and post-trigger IVUS imaging showing triggered luminal thrombus (yellow arrows) corresponding to the region of increased NIRF signal intensity on pre-trigger NIRF imaging in (B). (E) Ex vivo FRI of CLIO-CyAm7 corroborating in vivo 2D NIRF imaging. (F) Gross pathology of the resected aorta with 1.5cm black tissue markings for histological analysis and co-registration with in vivo imaging above. (G) Histogram of vessel diameter measured by cross sectional IVUS imaging. Red indicates atheroma without attached thrombus, yellow indicates atheroma with attached thrombus and blue indicates uninjured control aortic segments. The dashed line indicates the 5mm cutoff for exclusion of NIRF imaging data due to distance attenuation of the NIRF signal in large vessels. (H) In vivo 2D NIRF imaging showed significantly higher TBR in areas with atheroma, compared to uninjured segments of the aorta (peak TBR 2.86±1.82 and 1.55±0.65, p=0.001).

Figure 5. In vivo and microscopic analyses of nanoparticle deposition and triggered plaque thrombosis

(A) Cross sectional IVUS image of rabbit aorta with atheroma prior to pharmacologic triggering. (B,C) Post-triggering IVUS image corresponding to (A) demonstrating new luminal irregularity (yellow arrows and outline) consistent with new thrombus (segmented in (C)). (D,E) Corresponding histology of plaque with adherent thrombus. Carstairs’ staining of fibrin rich adherent thrombus (red). (F) RAM11+ macrophages are present at the surface below the thrombus. (G) Epifluorescence microscopy revealing increased CLIO-CyAm7 (red) at the plaque shoulder and underlying areas of thrombus. (H) Significantly higher CLIO-CyAm7 accumulation occurred in regions with thrombosis compared to atheroma without thrombosis (2.1±1.7%, n=34, and 1.5±1.9%, n=23, respectively, p=0.045). Scale bars: IVUS and low magnification histology = 1mm, high magnification histology = 100µm.