Single-Photon Emission Computed Tomography Imaging Using Formyl Peptide Receptor 1 Ligand Can Diagnose Aortic Aneurysms in a Mouse Model

Abstract

Background: Our previous studies showed that neutrophil infiltration and activation plays an important role in the pathogenesis of abdominal aortic aneurysms (AAA). However, there is a lack of noninvasive, inflammatory cell-specific molecular imaging methods to provide early diagnosis of AAA formation. Formyl peptide receptor 1 (FPR1) is rapidly upregulated on neutrophils during inflammation. Therefore, it is hypothesized that the use of cinnamoyl-F-(D)L-F-(D)L-F-K (cFLFLF), a PEGylated peptide ligand that binds FPR1 on activated neutrophils, would permit accurate and noninvasive diagnosis of AAA via single-photon emission computed tomography (SPECT) imaging.

Materials and methods: Male C57BL/6 (wild-type) mice were treated with topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat-inactivated elastase (control), and aortic diameter was measured by video micrometry. Comparative histology was performed on Day 14 to assess neutrophil infiltration in aortic tissue. We performed near-infrared fluorescence imaging using c-FLFLF-Cy7 probe on Days 7 and 14 postelastase treatment and measured fluorescence intensity ex vivo in excised aortic tissue. A separate group of animals were injected with ^99mTc-c-FLFLF 2 h before SPECT imaging on Day 14 using a SPECT/computed tomography/positron emission tomography trimodal scanner. Coexpression of neutrophils with c-FLFLF was also performed on aortic tissue by immunostaining on Day 14.

Results: Aortic diameter was significantly increased in the elastase group compared with controls on Days 7 and 14. Simultaneously, a marked increase in neutrophil infiltration and elastin degradation as well as decrease in smooth muscle integrity were observed in aortic tissue after elastase treatment compared with controls. Moreover, a significant increase in fluorescence intensity of c-FLFLF-Cy7 imaging probe was also observed in elastase-treated mice on Day 7 (approximately twofold increase) and Day 14 (approximately 2.5-fold increase) compared with respective controls. SPECT imaging demonstrated a multifold increase in signal intensity for ^99mTc-cFLFLF radiolabel probe in mice with AAA compared with controls on Day 14. Immunostaining of aortic tissue with c-FLFLF-Cy5 demonstrated a marked increase in coexpression with neutrophils in AAA compared with controls.

Conclusions: cFLFLF, a novel FPR1 ligand, enables quantifiable, noninvasive diagnosis and progression of AAAs. Clinical application of this inflammatory, cell-specific molecular probe using SPECT imaging may permit early diagnosis of AAA formation, enabling targeted therapeutic interventions and preventing impending aortic rupture.

Keywords: Abdominal aortic aneurysms; Aortic inflammation; Formyl peptide receptor; SPECT scans; Vascular imaging.

Figure 1.. Study design and phenotypic data.

(A) The schematic of the elastase treatment murine model of AAA. Infrarenal mouse aortas were treated with elastase or deactivated elastase (Control). Aortic diameter was measured on days 7 or 14, and tissue was harvested for further analysis. SPECT or fluorescence imaging was performed on days 7 and 14. (B) Elastase treated mice demonstrated a significant increase in aortic diameter compared with deactivated elastase treated mice on days 7 and 14, respectively. (C) Representative images of aortic phenotype in all groups. Aortic diameter increased in elastase treated groups compared to control groups. *P<0.0001 vs. Control Day 7; ^#P<0.0001 vs. Control Day 14; ns, not significant; n=6–15 mice/group.

Figure 2.. Increased neutrophil infiltration in AAA tissue.

Comparative histology performed on days 7 and 14 indicates that elastase-treated WT mice have a marked increase in neutrophil (PMN) infiltration, a decrease in smooth muscle cell α-actin (SM-αA) expression, as well as increase in elastic fiber disruption (Verhoeff-Van Gieson staining for elastin) compared to deactivated elastase-treated (control) mice, respectively. Quantification of histologic grading indicates a significant increase in neutrophil infiltration, decrease in SM-αA expression and increase in elastic fiber disruption in elastase-treated aortic tissue on days 7 and 14 compared to controls. Arrows indicate areas of immunostaining. *P<0.005 vs. Control Day 7; ^#P<0.0005 vs. Control Day 14; ns, not significant; n=5 mice/group.

Figure 3.. Increased uptake of c-FLFLF in AAA by fluorescence imaging of mice.

Near-infrared fluorescence imaging of c-FLFLF-Cy7 in live (shaved) anesthetized mice demonstrating aortic inflammation (elevated fluorescence) at the site of infrarenal AAA formation (see arrows) compared to controls on days 7 (A) and 14 (B). (C) Near-infrared fluorescence imaging of c-FLFLF-Cy7 in ex vivo aortic tissue demonstrating elevated fluorescence compared to controls on day14. (D) Fluorescence intensity (RFU; relative fluorescence units) in ex vivo aortic tissue was significantly increased on day 7 as well as on day 14 in elastase-treated mice (AAA) compared to respective controls. *P<0.05 vs. respective controls; ^#P<0.05 vs. Elastase Day 7; n=5 mice/group.

Figure 4.. Increased uptake of c-FLFLF in AAA by SPECT/CT imaging of mice.

(A) SPECT/CT imaging using ^99mTc-cFLFLF demonstrated an increase in c-FLFLF uptake (shown by arrows) in elastase-treated mice (AAA) compared to controls on day 14, signifying increased aortic inflammation relative to aneurysms. CT and SPECT scans were utilized to image the animal as it moved along the axis of rotation, allowing for combination of CT and SPECT images using stored offset parameters. (B) A significant increase in ^99mTc-cFLFLF radioactivity was observed in harvested aortic tissue from elastase-treated mice compared to controls. *P<0.05 vs. control; n=4–5 mice/group.

Figure 5.. Co-expression of c-FLFLF with PMNs in AAA tissue.

(A) Immunostaining of murine aortic tissue demonstrates increased c-FLFLF-Cy5 (red) expression in elastase-treated WT mice (bottom panel) compared to controls (top panel) on day 14. A marked increase in neutrophil (Ly6G-FITC; green) expression is also observed in elastase-treated mice compared to control. Moreover, the c-FLFLF expression is co-localized with neutrophils (Ly6G-FITC) indicating the neutrophil-specificity of the c-FLFLF probe. Arrows indicate specific and increased areas of c-FLFLF-Cy5 and Ly6G-FITC expression. (B) Quantification of same normalized areas of imaging demonstrates increased fluorescence intensity of c-FLFLF-Cy5 and Ly6G-FITC. ns, not significant; *P<0.05 vs. respective controls; n=5 mice/group.