CCR2 Positron Emission Tomography for the Assessment of Abdominal Aortic Aneurysm Inflammation and Rupture Prediction

Abstract

Background: The monocyte chemoattractant protein-1/CCR2 (chemokine receptor 2) 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, using a targeted positron emission tomography radiotracer (⁶⁴Cu-DOTA-ECL1i).

Methods: AAAs were generated in Sprague-Dawley rats by exposing the infrarenal, intraluminal aorta to PPE (porcine pancreatic elastase) under pressure to induce aneurysmal degeneration. Heat-inactivated PPE was used to generate a sham operative control. Rat AAA rupture was stimulated by the administration of β-aminopropionitrile, a lysyl oxidase inhibitor. Biodistribution was performed in wild-type rats at 1 hour post tail vein injection of ⁶⁴Cu-DOTA-ECL1i. Dynamic positron emission tomography/computed tomography imaging was performed 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 computed tomography. At day 7 post-AAA induction, the radiotracer uptake (standardized uptake value [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 nonruptured AAAs (SUV=0.73±0.11). Histopathologic 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 positron emission tomography is a promising new biomarker for the noninvasive assessment of AAA inflammation that may aid in associated rupture prediction.

Keywords: abdominal aortic aneurysm; autoradiography; cc chemokine receptor 2; molecular imaging; positron emission tomography; rupture.

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.

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.

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.

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.

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.