CCR2 positron emission tomography for the assessment of abdominal aortic aneurysm inflammation and rupture prediction

Abstract

BACKGROUND:

The monocyte chemoattractant protein-1/chemokine receptor 2 (CCR2) axis plays an important role in abdominal aortic aneurysm (AAA) pathogenesis, with effects on disease progression and anatomic stability. We assessed the expression of CCR2 in a rodent model and human tissues, utilizing a targeted positron emission tomography (PET) radiotracer (⁶⁴Cu-DOTA-ECL1i).

METHODS:

AAAs were generated in Sprague-Dawley rats by exposing the infrarenal, intraluminal aorta to porcine pancreatic elastase (PPE) under pressure to induce aneurysmal degeneration. Heat-inactivated PPE was utilized to generate a sham operative control. Rat AAA rupture was stimulated by the administration of β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor. Biodistribution was performed in wild-type rats at 1 hour post tail vein injection of ⁶⁴Cu-DOTA-ECL1i. Dynamic PET/computed tomography (CT) imaging was carried out in rats to determine the in vivo distribution of radiotracer.

RESULTS:

Biodistribution showed fast renal clearance. The localization of radiotracer uptake in AAA was verified with high-resolution CT. At day 7 post AAA induction, the radiotracer uptake (SUV= 0.91±0.25) was approximately twice that of sham-controls (SUV = 0.47 ± 0.10, p<0.01). At 14 days post AAA induction, radiotracer uptake by either group did not significantly change (AAA SUV = 0.86 ± 0.17 and sham-control SUV = 0.46±0.10), independent of variations in aortic diameter. Competitive CCR2 receptor blocking significantly decreased AAA uptake (SUV=0.42±0.09). Tracer uptake in AAAs that subsequently ruptured (SUV=1.31±0.14, p<0.005) demonstrated uptake nearly twice that of non-ruptured AAAs (SUV= 0.73±0.11). Histopathological characterization of rat and human AAA tissues obtained from surgery revealed increased expression of CCR2 that was co-localized with CD68+ macrophages. Ex vivo autoradiography demonstrated specific binding of ⁶⁴Cu-DOTA-ECL1i to CCR2 in both rat and human aortic tissues.

CONCLUSIONS:

CCR2 PET is a promising new biomarker for the non-invasive assessment of AAA inflammation that may aid in associated rupture prediction.

INTRODUCTION

Abdominal aortic aneurysms (AAAs) represent a life-threatening degenerative vascular disease characterized by transmural aortic macrophage infiltration, elastin degradation, and the reduction of vascular smooth muscle cell (VSMCs) content.^{1, 2} Aneurysms typically remain asymptomatic until they rupture, leading to high mortality and a substantial burden on the health care system.^{3, 4} The current clinical approach for screening and surveillance is almost entirely based on anatomic measurements, without assessing the cellular or molecular processes associated with aneurysm expansion and rupture. Imaging-based prediction of aneurysm rupture, remains a significant clinical challenge. Twenty percent of ruptured AAAs are less than 5 cm in diameter,⁵ and 40% of AAAs 7–10 cm do not rupture.⁶ Though some molecular probes have been developed to image specific biomarkers expressed by AAAs, including matrix metalloproteinases (MMPs) and integrin α_vβ₃ in preclinical research,^7–12 there is an unmet clinical need to develop translatable molecular probes to identify those AAAs that are prone to expand and/or rupture and deliver patient specific medical and/or surgical therapy.⁹

The pathological features of AAA vary based on disease severity, but atherosclerosis, extracellular matrix (ECM) remodeling, loss of VSMCs and increased inflammatory cell activity are generally present.¹³ It is known that inflammatory cells play crucial roles in AAA pathogenesis by accumulating in the aortic wall, releasing inflammatory factors and activating proteases that promote matrix degradation.¹⁴ Specifically, monocytes and macrophages have been widely studied both experimentally and clinically, due to their critical roles in all stages of AAA formation.^15–17 Among biomarkers expressed on monocytes/macrophages, chemokine receptor type 2 (CCR2) is of particular interest, due to its role in mediating leukocyte trafficking to the site of inflammation in the arterial wall following injury.¹⁸ A reduction of proinflammatory monocytes, due to treatment by pharmacological intervention or genetic depletion of CCR2, significantly attenuated the dilatation and rupture of aneurysms in ApoE^−/− mice,^18–24 suggesting the potential of CCR2 as a theranostic biomarker for AAA.

We have developed a positron emission tomography (PET) imaging agent ⁶⁴Cu-DOTA-ECL1i for targeting CCR2 and demonstrated its utility in multiple animal models.^25–29 Herein, we have characterized the imaging sensitivity and specificity of ⁶⁴Cu-DOTA-ECl1i in a rat AAA model, over the acute and chronic phases of aneurysm development. We have validated the expression of CCR2 in human atherosclerotic and aneurysmal aortic tissues. Moreover, we have assessed the potential of CCR2 PET to predict rat AAA rupture.^{30, 31}

METHODS

Animal models

The data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure. The study was approved by the Institutional Animal Care and Use Committee at Washington University. Male Sprague-Dawley rats (200 to 300 g) were obtained from Charles River Laboratories (Wilmington, MA, USA) and utilized for all experiments. Animal anesthesia was performed with a mixture of approximately 1.5% isoflurane and oxygen for all procedures. The core body temperature was maintained with a heating pad (37 °C).

AAA and sham-control models

AAAs (n = 12) were established utilizing active PPE (12 U/mL), while control animals (n=9) were exposed to heat-inactivated PPE (12 U/mL, heated at 90°C for 45 min) as previously described.³⁰ After ventral abdominal wall incision, a customized polyethylene catheter (Braintree Scientific, Braintree, MA, USA) was introduced through an aortotomy, and elastase was introduced into the isolated aortic segment for 30 min. The exposed aortic segment was dilated to a maximal diameter, and constant pressure was maintained with the use of a syringe pump. Using a video micrometer, the maximum aortic diameter was measured. Both AAA and sham-control rats were scanned with PET/CT at day 7 and day 14 post-induction (Figure S1).

AAA model stimulated to rupture

Starting 3 days prior to elastase exposure and daily thereafter, male rats (n = 13) underwent β-aminopropionitrile (BAPN) administration through drinking water (0.3% BAPN in water. Rats were approximately 250 g and drank 25 mL water/day, leading to intake of ~300 mg BAPN/kg/day).³² Elastase (12 U/mL) was introduced into the isolated aortic segment for 30 minutes as abovementioned. The growth of AAA was monitored during the 14-day study. AAA rats ruptured over the course of the study were included in the ruptured AAA (RAAA) group (n = 7); whereas those animals that did not rupture by day 14 post elastase exposure (PEE) were referred to as the non-ruptured AAA group (NRAAA, n = 6).

Noninvasive aortic diameter measurements

Utilizing ultrasound (US, 12 MHz Zonare, Mountain View, CA, USA), the maximum aortic diameter was measured noninvasively. Percentage increases in aortic diameter were determined considering the baseline intraluminal aortic diameter and the maximum aortic diameter at days 7 and 14 post PEE. An aortic aneurysm was defined by a >100% increase in the aortic diameter compared to pretreatment measurements.³⁰

Synthesis and radiolabeling of DOTA-ECL1i

The ECL1i peptide (LGTFLKC) was synthesized from D-form amino acids by CPC Scientific (Sunnyvale, CA). DOTA-ECL1i was prepared following our previous report.²⁷ Copper-64 (⁶⁴Cu, t_1/2=12.7h) was produced by the Washington University Cyclotron Facility. The DOTA-ECL1i conjugate was radiolabeled with ⁶⁴CuCl₂ (⁶⁴Cu-DOTA-ECL1) as described and the radiochemical purity was determined by radio-HPLC.²⁷

Biodistribution

Wild type (WT) male Sprague-Dawley rats (200–225g, 8 weeks old, n=4/group) were injected with ⁶⁴Cu-DOTA-ECL1i via tail vein (100 μL, 1.11 MBq per rat). At 1 h post injection, rats were euthanized and organs of interested were collected, weighed, and assayed by a gamma counter (Beckman, Brea, CA) as described.²⁹ Standards were prepared and measured in parallel to calculate the percentage of the injected dose per gram of tissue (%ID/g).

Small Animal PET/CT imaging and image analysis

Dynamic PET scan and corresponding computed tomography (CT) images were obtained using Inveon MM PET/CT (Siemens, Malvern, PA) at 45–60 min after a tail vein injection of ⁶⁴Cu-DOTA-ECL1i (11.1 MBq per rat) to minimize the effect of blood retention on AAA uptake. To localize tracer uptake, a CT contrast agent (1.0 mL, eXIA 160XL, Binitio, Canada) was administrated via tail vein after PET imaging. Contrast-enhanced CT (Bin of 2, 90 mm axial FOV, 60kV, 500μA, 500ms exposure time, 10 ms settle time, no magnification, pixel size: 80–100μm) was performed. The organ uptake was calculated as standardized uptake value (SUV) in three-dimensional regions of interest (ROIs) from PET images without correction for partial volume effect using Inveon Research Workplace software (Siemens).³³ Competitive PET blocking studies were performed in the AAA rat model with co-injection of non-radiolabeled ECL1i and ⁶⁴Cu-DOTA-ECL1i (ECL1i:⁶⁴Cu-DOTA-ECL1i molar ratio = 500:1), followed by a 45–60 minute dynamic scan. Dynamic (0–90 min)¹⁸F-FDG (41.1 MBq per rat) PET was also performed in AAA rats at day 7 PEE.

Histology and immunofluorescent staining of rat AAA tissue sections

Formalin fixed, paraffin embedded sections were deparaffinized in xylenes and rehydrated through a series of graded alcohols. Tissues were processed for antigen retrieval by boiling in Diva Decloaker (pH 6.2, Biocare Medical). They were blocked in 10% donkey serum for 1 h to prevent nonspecific binding. The sections were then incubated overnight at 4°C in primary antibody (anti-CCR2, Novus Biologicals, 1:100 and anti-CD68, Biorad, 1:100) or control IgG (anti-rabbit and anti-mouse IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in phosphate-buffered saline (PBS), mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) stains were performed on serial sections to analyze morphology and severity of the AAA tissues. The infiltrated inflammatory cells were counted by a clinical pathologist at 3 randomly selected 20x region-of-interest of AAA and sham-control tissue slides (n=4/group). Pentachrome stain was also carried out to characterize the collagen content of AAA and sham-control tissues.

Whole tissue immunofluorescent staining of rat AAAs

After harvest, whole rat aortas were washed in PBS and then fixed in 30% sucrose mixed with 4% PFA for 2–3 h. Tissues were then blocked in 1% BSA with 1% Triton X-100 for 4 h to prevent nonspecific binding, and whole tissues were then incubated for 48 h at 4°C in primary antibody (anti-CCR2, Novus Bio, 1:50 and anti-CD68, Bio-Rad, 1:100) or control IgG (anti-mouse and anti-rabbit IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 24 h at 4°C, and tissues were washed in PBS and incubated with DAPI (1:700 in PBS from Sigma). Tissues were then cleared in 70% ethanol, followed by 100% ethanol overnight. Whole tissues were then mounted in aqueous mounting media and imaged using a Zeiss LSM 880 II Airyscan FAST confocal microscope.

Reverse transcription-polymerase chain reaction (RT-PCR) of human AAA tissues

Human AAA tissues were obtained under the approval of the institutional review board. RNA isolated from excised human AAA specimens was used for real time RT-PCR. Tissue RNA was isolated using Nucleospin RNA kits (Macherey-Nagel; Bethlehem, PA) per the manufacturer’s instruction. Reverse transcription reactions used 1 μg of total RNA, random hexamer priming, and Superscript II reverse transcriptase (Invitrogen). Expression of CCR2 and RNA polymerase II subunit A (POLR2A) were determined using Taqman assays (Invitrogen) and an EcoTM Real-Time PCR System (Illumina, San Diego, CA) in duplicate in 48-well plates. PCR cycling conditions were as follows: 50°C for 2 min, 95°C for 21 s, and 60°C for 20 s. POLR2A expression was used as a comparator using ΔΔ Ct calculations.

Histology and immunofluorescent staining of human AAA tissues

After directly placing in saline upon excision of the AAA during surgery, human specimens were fixed in 10% formalin, embedded in OCT compound, frozen, and sectioned. Frozen sections were hydrated in PBS, and blocked in 10% donkey serum for 1 h to prevent nonspecific binding. The sections were then incubated overnight at 4°C in primary antibody (anti-CCR2, Abcam, 1:400 and anti-CD68, Millipore Sigma, 1:100) or control IgG (anti-mouse and anti-rabbit IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in PBS, mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) and Verhoeff-Van Gieson (VVG) stains were performed on serial sections to analyze morphology and severity of the AAA tissues.

Autoradiography of rat and human AAA tissue

For AAA and sham-control rats, after PET imaging at day 14 PEE, the thoracic and abdominal aortas and the psoas muscle were collected after rats were euthanized and infused with saline. The aortic tissue was sliced longitudinally and fixed in paraformaldehyde for 2 h, followed by 2 × 5-minute rinses with 0.9% sodium chloride. All tissues were placed on a charged phosphor screen and exposed overnight.

De-identified paraffin-embedded human AAA tissue was acquired from the Vascular Surgery Department Bio Bank under the approval from the Human Research Protection Office of Washington University. Slides were deparaffinized and rehydrated through a series of graded alcohols. Tissues were processed for antigen retrieval by boiling in citrate-based Unmasking Solution (pH 6, Vector Laboratories) and blocking for non-specific binding with 1% bovine serum albumin in 0.1% phosphate buffered saline plus Tween. The slides were submerged in a Coplin jar containing 1.11 MBq/mL of ⁶⁴Cu-DOTA-ECL1i in MilliQ water and incubated for 15 minutes with shaking. The tracer was decanted and the slides were rinsed 3 times with MilliQ water and exposed overnight on a charged phosphor screen. All autoradiography was analyzed with a GE Typhoon FLA 9500 Variable Mode Laser Scanner at 50 micron resolution. ImageJ was used for the quantification of ⁶⁴Cu-DOTA-ECL1i uptake in AAA tissue samples. A region of interest was drawn around the entire tissue section. Mean pixel intensity was compared between AAA samples and sham-controls or the blocked samples incubated with an excess of ECL1i.

Statistical Analysis

Data are presented as the mean ± standard deviation. Group comparisons were performed using the one-way ANOVA. The significance level threshold in all tests was p ≤ 0.05. The D’Agostino & Pearson omnibus normality test was performed. GraphPad Prism v. 6.04 (La Jolla, CA) was used for all statistical analyses.

Results

Biodistribution of ⁶⁴Cu-DOTA-ECL1i in rats

The in vivo pharmacokinetic evaluation of ⁶⁴Cu-DOTA-ECL1i performed in WT rats at 1 h post injection (p.i.) via tail vein showed fast blood clearance and efficient renal excretion (Figure 1). At 1 h p.i., there was less than 0.2%ID/g tracer retained in any of the organs, with the exception of the kidneys due to renal clearance, consistent with a previous report.²⁷ The uptake ratios of aorta/blood and aorta/heart were both approximately 1, indicating that the accumulation of tracer in the aorta was mainly due to blood retention.

Figure 1.

Biodistribution of ⁶⁴Cu-DOTA-ECL1i in wild type rats (n=4/group). A. Biodistribution of ⁶⁴Cu-DOTA-ECL1i demonstrated rapid renal clearance and low blood pool retention at 1 h post injection via tail vein. B. Uptake ratios demonstrated comparable retention of radiotracer in the blood, heart, and aorta.

Rat AAA ⁶⁴Cu-DOTA-ECL1i PET imaging

The rat AAA model and sham-controls were established as previously reported.^{30, 31} CT contrast agent was used to define the aortic dilation and identify the intraluminal space using high-resolution CT. At day 14 PEE, ⁶⁴Cu-DOTA-ECL1i PET signal specifically localized within the AAA (Figure 2). The high kidney and bladder retention of radiotracer on PET image was consistent with the data acquired in the biodistribution study.

Figure 2.

Contrast enhanced localization of ⁶⁴Cu-DOTA-ECL1i in the abdominal aortic aneurysm of a rat. High resolution CT images in various orientations with contrast enhancement identifying the AAA (green arrow). PET, and PET/CT fused images demonstrated tracer uptake at the intraluminal boundaries of the AAA. HU: Hountsfield unit.

Time course studies were performed in AAA and sham-control rats at day 7 and 14 PEE to follow aneurysm progression. PET images and quantification demonstrated an approximate 2-fold greater accumulation of ⁶⁴Cu-DOTA-ECL1i by AAAs at both day 7 (SUV= 0.91±0.25, n=12) and day 14 PEE (SUV= 0.86±0.17, n=9) compared to sham-control aortas at the corresponding time points (day 7 SUV= 0.47±0.10, n=4, p=0.0018; day 14 SUV= 0.46 ± 0.10, n=4, p=0.0085) (Figure 3A, B). Competitive receptor blocking study using non-radioactive DOTA-ECL1i performed at day 14 PEE showed significantly decreased radiotracer uptake by AAA (SUV=0.42±0.09, n=4, p=0.0100) (Figure 3A), demonstrating targeting specificity, which was consistent with our previous PET studies using blockade and CCR2 knock-out mice.^{26, 27, 34} In WT rat abdominal aorta, little ⁶⁴Cu-DOTA-ECL1i accumulation (SUV = 0.29±0.02, n=3) was determined, indicating low nonspecific retention (Figure 3A). The tracer uptake presented as %injected dose also showed significantly higher uptake in AAA rats than those in sham-controls and blocking studies (Figure S2). ¹⁸F-FDG PET/CT performed at day 7 PEE in AAA rats revealed comparable uptake (SUV = 0.84±0.42, n=3) to that of ⁶⁴Cu-DOTA-ECL1i, which was also consistent with our previous report in the same rat AAA model (Figure S3).^{30, 31} The aorta/heart uptake ratios were also calculated to further assess AAA targeting specificity considering radiotracer retention in blood. As shown in Figure 3C, the ratios in AAAs were significantly (p<0.0001) greater than those of sham-control rats. Moreover, DOTA-ECL1i blockade substantially (p<0.0001, n=4) decreased the aorta/heart uptake ratio at day 14 to a level similar to that observed in sham-control rats. In WT rats, the uptake ratio was comparable to the results determined from biodistribution study, confirming little non-specific tracer retention in the rat abdominal aorta. The aorta/muscle ratios of ⁶⁴Cu-DOTA-ECL1i uptake in the rat models were also assessed. Similar to the trends demonstrated in Figure 3C, ⁶⁴Cu-DOTA-ECL1i uptake ratios were significantly higher in AAA rats than those acquired in sham-control rats, as well as AAA rats that received blockade or WT rats (Figure 3D).

Figure 3. ⁶⁴Cu-DOTA-ECL1i PET/CT of rat AAA.

A, Representative PET/CT images at day 7 and 14 post-elastase exposure showed specific and intensive detection of aneurysm (yellow arrow) compared to the low trace accumulations in the sham-control rats. Competitive receptor blocking studies using excess non-radiolabeled ECL1i peptide significantly reduced the tracer uptake at day 14 PEE. PET/CT image in WT rat without elastase exposure showed little tracer retention in abdominal aorta. B. Quantitative tracer uptake of abdominal aorta, C. abdominal aorta/heart uptake ratios, D. abdominal aorta/muscle uptake ratios, E. abdominal aorta/kidney uptake ratios of ⁶⁴Cu-DOTA-ECL1i in AAA and sham-control rats at day 7 and 14 PEE. F. Ex vivo autoradiography of ⁶⁴Cu-DOTA-ECL1i in thoracic aortas, abdominal aortas and psoas muscle of AAA and sham-control rats. G. Confocal images of immunofluorescent staining of whole abdominal aortas of AAA and sham-control rats at day 14 PEE. H. H&E staining of abdominal aortas (cross section of tissue slides) from AAA and sham-control rats at day 14 PEE. I. Percent increase of aortic diameters of AAA and sham-control rats at day 7 and 14 PEE determined by ultrasound.

Ex vivo autoradiography demonstrated strong tracer binding to abdominal aorta but low retention in thoracic aorta and psoas muscle collected from AAA rats (Figure 3E). For the sham-control rats, radiotracer retention was measured in abdominal aorta but the uptake was much less than that observed in AAA rats. Additionally, little radiotracer uptake was observed in the thoracic aorta and muscle, consistent with imaging results in the AAA rat.

Immunofluorescent staining of the pre-fixed whole abdominal aorta showed CD68+ cells throughout the abdominal aorta of AAA rat with many cells also positive for CCR2, particularly within the segment of greatest aortic dilatation (Figure 3F). Furthermore, these regions of increased CD68 and CCR2 expression corresponded with increased radiotracer uptake observed with autoradiography. Sham-control aortas demonstrated less CD68 and CCR2 expression, compared to those of AAAs. For both groups, the radiotracer binding profiles on autoradiography were comparable to those of CCR2 immunofluorescent staining, indicating the specific binding of ⁶⁴Cu-DOTA-ECL1i to upregulated CCR2 in the rat AAA.

Compared to sham-controls, H&E stained sections of AAAs collected on day 14 PEE demonstrated dilated aortas, with disorganized VSMCs, disrupted elastic laminae, and increased inflammatory cells and deposition of collagen in the tunica intima and media. In addition to large mural thrombi, acute hemorrhage was also noted in the tunica media. These histopathological features of rat AAAs largely recapitulated that of human AAAs, lending support to use of the rat model for imaging AAA associated inflammation (Figure 3G, Figure S4).

Moreover, the diameters of rat abdominal aortas measured by ultrasound on day 7 PEE demonstrated 202 ± 68 % (n=10) increases by AAAs, which further increased to 250 ± 34 % (n=4) at day 14, while the sham-controls showed significantly less increase at day 7 (96 ± 40 %, n=10, p=0.0007) and day 14 (94 ± 48%, n=5, p=0.0009) (Figure 3H). At day 7 PEE, H&E stained sections from AAAs revealed prominent inflammatory reactions in all three layers of the vessel (tunica intima, media and adventitia) as well as the development of mural thrombi, suggesting disturbed blood flow and endothelial cell damage. Day 7 whole-mount immunofluorescent staining of the AAAs demonstrated globally increased CD68 and CCR2 expression, particularly at the level of greatest aortic dilatation (Figure S5).

CCR2 PET imaging in a rupture-prone rat AAA model

In contrast to the significant tracer uptake in NRAAA rats, the RAAA rat revealed a much stronger PET signal within the aneurysm at day 6 PEE (Figure 4). Quantitative uptake analysis showed that the accumulation of ⁶⁴Cu-DOTA-ECL1i in RAAAs (SUV=1.31±0.14, n=7) was significantly (p<0.0001) greater than that of NRAAAs (SUV=0.73±0.11, n=6) (Figure 4 A,B). Moreover, the aorta/heart (5.21±0.72, n=7) and aorta/muscle (7.04±1.09, n=7) uptake ratios associated with RAAAs was almost double those of NRAAAs (3.10±0.32, 4.23±0.70, n=7, respectively, Figure 4 C,D), demonstrating the potential of ⁶⁴Cu-DOTA-ECL1i to assess aneurysm vulnerability. Moreover, the similar aortic diameters of RAAAs and NRAAAs supports a mechanism of AAA rupture that is driven by inflammation, independent of aortic dilatation.

Figure 4. Comparison of ⁶⁴Cu-DOTA-ECL1i PET/CT in NRAAA and RAAA rats.

A. Representative PET/CT images at day 6 post-elastase exposure showed more intensive signal in RAAA rats (yellow arrow) compared to the NRAAA rats. B. Quantitative uptake, C. abdominal aorta/heart uptake ratios, D. abdominal aorta/muscle uptake ratios of ⁶⁴Cu-DOTA-ECL1i in NRAAA and RAAA rats at day 6 post-elastase exposure. E. Percent increase of aortic diameters of NRAAA and RAAA at day 6 PEE determined by ultrasound.

Human tissue validation of CCR2 as a biomarker for AAA

Human AAA samples were examined histologically and immunophenotypically. In cases with moderate to severe degrees of aneurysmal formation, typical pathological findings included loss of VSMCs, disruption of elastin lamina, increased collagen deposit in the tunica media, increased inflammatory infiltrates, and atheromatous changes. Mural thrombi were often observed (Figure 5A, B, D and E). CD68 immunofluorescent stains demonstrated CD68+ macrophages throughout the vessel wall, from mural thrombi, neointima, tunica media, to adventitial layers. CCR2 staining also revealed elevated expression in a pattern similar to CD68 which was demonstrated by their co-localization in the majority of CD68+ macrophages (Figure 5C, F–I).

Figure 5. Ex vivo characterization of representative human abdominal aortic aneurysm tissue.

A. H&E B. VVG staining of human AAA specimen with moderate severity. C. Immunofluorescent staining of CD68 and CCR2 using adjacent slide of same specimen for Figure 5A and 5B. D-F. Higher magnifications of HE stained, VVG stained, and immunofluorescent stained human AAA tissue. The corresponding area was annotated by the boxes in figure 5A-C. G-I. Separate channels for DAPI (G), CD68 (H), and CCR2 (I) staining of AAA specimen from figure 5C. J. Ex vivo autoradiography of human AAA specimen incubated with ⁶⁴Cu-DOTA-ECL1i revealed intensive but heterogeneous distribution of tracer binding. K. Competitive blocking assay utilizing excess non-radiolabeled ECL1i demonstrating decreased radiotracer binding. L. Quantitative RT-PCR analysis of human AAA tissues demonstrated CCR2 was the highest among the 7 chemokine receptors (n=4). * p=0.0003 for CCR1 vs. CCR2, p<0.0001 for CCR3, CCR4, CCR5, CCR8 or CXCR4 vs. CCR2.

Autoradiography performed on the consecutive cut of same human AAA specimens showed a strong but heterogeneous tracer distribution (Figure 5J) in a pattern similar to that of CCR2 immunostaining (Figure 5I). Specifically, CD68+ and CCR2+ macrophages were detected in the lymph nodes and fibrous tissue within adventitial and the organizing mural thrombi and neointima on the luminal surface (Figure S6) where intensive radiotracer binding was also observed, suggesting the specific binding of ⁶⁴Cu-DOTA-ECL1i to CCR2+ macrophage. Furthermore, the significantly decreased radiotracer binding by competitive receptor blocking on human AAA tissue confirmed the binding specificity (Figure 5K).

To further verify the potential of CCR2 as a biomarker for human AAA, RNA was extracted from freshly collected human AAA tissues (n=4) for RT-PCR analysis. As shown in Figure 5L, of the 7 chemokine receptors analyzed, CCR2 was significantly (p=0.0003 for CCR1, p<0.0001 for CCR3, CCR4, CCR5, CCR8 and CXCR4) higher than the other 6 targets, indicating the potential of CCR2 imaging of human AAA.

Discussion

Monocytes and macrophages play a critical role in AAA development by driving the process of aortic wall remodeling^{13, 14, 35, 36} and have been used as diagnostic biomarkers for AAA imaging.^{10, 37} Of various biomarkers expressed by monocytes/macrophages, the CCR2/MCP-1 axis plays an important role in monocyte recruitment and activation, promotion of vascular inflammation, as well as ECM remodeling and aneurysm stability, suggesting the potential of CCR2 as a biomarker for AAA management.^{13, 14, 38, 39} Previously, we have shown the specific targeting of CCR2+ cells using ⁶⁴Cu-DOTA-ECL1i in multiple inflammatory animal models.^25–29 Herein, we reported the sensitive and specific detection of CCR2+ cells in a rat AAA model utilizing ⁶⁴Cu-DOTA-ECL1i and assessed its application in a rat AAA rupture model. The specific binding of this tracer to CCR2 up-regulated in ex vivo human AAA specimens supports the potential application of utilizing ⁶⁴Cu-DOTA-ECL1i for human AAA imaging and therapy.

In order to monitor the dynamic variation of a specific biomarker over the course of AAA progression and/or assess a treatment response, adequate and reliable target-to-background contrast is necessary. Consistent with our previous pharmacokinetic data in mice, we demonstrated rapid blood clearance and renal excretion of ⁶⁴Cu-DOTA-ECL1i in WT rats, which led to a low background in aorta to enhance imaging contrast.⁴⁰ Compared to mouse AAA models, our rat AAA model afforded a larger aneurysm that allowed for improved anatomic resolution and localization of PET signal. It is known that for vascular imaging, not only the sensitivity and specificity, but also the fast blood clearance and low residence of a tracer are important to enhance the imaging contrast in order to delineate the status and progression of disease.⁴⁰ In the current study, elevated CCR2 expression in AAAs led to significantly higher radiotracer uptake compared to sham-control aortas, suggesting CCR2 imaging sensitivity, which was further verified by ex vivo autoradiography. In contrast to the comparable radiotracer uptake in the aorta and heart of WT rats as determined in both biodistribution and PET imaging studies, the aorta/heart SUV ratios of AAAs was approximately 2.5 times greater, suggesting the aneurysm targeting specificity, which was further confirmed by the significantly decreased radiotracer uptake in AAAs caused by the competitive blockade.

An unmet clinical need for AAA management is the ability to identify patients at risk of aneurysm growth and/or rupture.⁶ Recent studies in AAA patients utilizing molecular imaging have highlighted the potential of macrophage driven aortic wall inflammation as an effective approach to track aneurysm expansion, rupture, and/or the need for surgical repair.^{6, 41} Herein, ⁶⁴Cu-DOTA-ECL1i uptake by RAAAs was approximately twice that of NRAAAs, independent of comparable aortic diameters of the two groups. This suggested the further evaluation of ⁶⁴Cu-DOTA-ECL1i PET in rupture-prone preclinical AAA models as a non-invasive, molecular imaging approach to assess CCR2+ cells variation during AAA growth and expansion is warranted.

As previously reported, human specimens are the most appropriate platform to assess the translational potential of molecular probes.^{42, 43} In our studies, we showed that the representative human AAA tissue contains CCR2⁺/CD68⁺ macrophages in all layers of the aorta. Ex vivo autoradiography using ⁶⁴Cu-DOTA-ECL1i demonstrated a comparable profile to the immunostaining. The result is supportive of ⁶⁴Cu-DOTA-ECL1i for translational human AAA imaging.

Our study has several limitations. CCR2 was detected in both rat and human aneurysm specimens that were electively collected. Ruptured rat and human AAA tissues were not analyzed. Moreover, the rat AAA rupture model utilized for this study is driven by the acute, subacute, as well as chronic phases of inflammation contributing to the associated AAA pathophysiology. Though a chronic model of AAA development and rupture has been proposed, the associated AAA rupture is far less predictable, and rupture occurs during all phases of inflammation as well.³² A more clinically relevant animal model is necessary to further assess the potential of CCR2 PET for AAA imaging and rupture prediction. Another limitation is that the PET radiotracer uptake was not corrected for partial volume effect. However, the uptake in RAAA rats was nearly double that of NRAAA rats ensuring the targeting specificity of ⁶⁴Cu-DOTA-ECL1i given the comparable sizes of AAAs in the two groups. Additionally, the human tissue validation was done on limited AAA specimens; an array of human AAA tissues containing various level of CCR2⁺/CD68⁺ macrophages are needed to establish the correlation between ⁶⁴Cu-DOTA-ECL1i binding and immunostaining.

In summary, the up-regulation of CCR2 in a rat AAA model stimulated to rupture and by human AAAs demonstrated its potential as a target for molecular imaging and perhaps therapy in AAA. ⁶⁴Cu-DOTA-ECL1i PET showed sensitive and specific detection of CCR2 in both rat and human AAA tissue. Given that ⁶⁴Cu-DOTA-ECL1i is currently undergoing clinical evaluation in patients for the study of other disease processes,⁴⁴ the results of this study support the translational study of ⁶⁴Cu-DOTA-ECL1i PET imaging of AAA patients.

CLINICAL PERSPECTIVE.

AAA is a leading cause of death in the aging population. Aortic dilatation is nonlinear and unpredictable, and no medical therapy exists for the management of AAAs. A reliable biomarker has not been identified that may aid in the surveillance of AAAs for associated growth and/or rupture prediction. Pathogenesis of AAA demonstrated that pro-inflammatory monocytes/macrophages are major drivers of AAA progression and ultimate rupture. C-C motif chemokine receptor 2 (CCR2) is a biomarker overexpressed by pro-inflammatory leukocytes to mediate their trafficking to inflammatory sites. Herein, we confirmed the over-expression of CCR2 in a rat AAA model and human AAA specimens, both co-localized with macrophages. We demonstrated the sensitive and specific detection of CCR2 utilizing ⁶⁴Cu-DOTA-ECL1i, a CCR2 targeted PET radiotracer. Elevated radiotracer uptake by rupture-prone AAAs, as well as specific binding by human AAA tissue, suggests that ⁶⁴Cu-DOTA-ECL1i might be utilized for AAA imaging in humans.

Non-standard Abbreviations and Acronyms

¹⁸F-FDG

¹⁸F-fluorodeoxyglucose

AAA

abdominal aortic aneurysms

ApoE^−/−

apolipoprotein E knock-out

BAPN

β-aminopropionitrile

CCR2

C-C chemokine receptor type 2

CT

computed tomography

DOTA

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

ECL1i

extracellular loop 1 inverso

ECM

extracellular matrix

HPLC

high performance liquid chromatography

MMPs

matrix metalloproteinases

PEE

post elastase exposure

PET

positron emission tomography

PPE

porcine pancreatic elastase

ROI

region of interest

SUV

standardized uptake value

VSMCs

vascular smooth muscle cells

WT

wild type