Shannon Alexander H, Chordia Mahendra D, Spinosa Michael D, Su Gang, Ladd Zachary, Pan Dongfeng, Upchurch Gilbert R, Sharma Ashish K
Department of Surgery, University of Virginia, Charlottesville, Virginia.
Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, Virginia.
J Surg Res. 2020 Jul;251:239-247. doi: 10.1016/j.jss.2020.01.028. Epub 2020 Mar 12.
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.
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 Tc-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.
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 Tc-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.
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.
我们之前的研究表明，中性粒细胞浸润和激活在腹主动脉瘤（AAA）的发病机制中起重要作用。然而，缺乏用于AAA形成早期诊断的非侵入性、炎症细胞特异性分子成像方法。甲酰肽受体1（FPR1）在炎症过程中在中性粒细胞上迅速上调。因此，有人推测，使用肉桂酰 - F -（D）L - F -（D）L - F - K（cFLFLF），一种与活化中性粒细胞上的FPR1结合的聚乙二醇化肽配体，将能够通过单光子发射计算机断层扫描（SPECT）成像对AAA进行准确的非侵入性诊断。
雄性C57BL/6（野生型）小鼠局部用弹性蛋白酶（0.4 U/mL 1型猪胰弹性蛋白酶）或热灭活弹性蛋白酶（对照）处理，通过视频显微镜测量主动脉直径。在第14天进行比较组织学检查以评估主动脉组织中的中性粒细胞浸润。在弹性蛋白酶处理后的第7天和第14天，使用c - FLFLF - Cy7探针进行近红外荧光成像，并在切除的主动脉组织中离体测量荧光强度。在第14天，使用SPECT/计算机断层扫描/正电子发射断层扫描三模态扫描仪，在SPECT成像前2小时给另一组动物注射Tc - c - FLFLF。在第14天，还通过免疫染色对主动脉组织进行中性粒细胞与c - FLFLF的共表达检测。
与对照组相比，弹性蛋白酶组在第7天和第14天主动脉直径显著增加。同时，与对照组相比，弹性蛋白酶处理后主动脉组织中观察到中性粒细胞浸润和弹性蛋白降解明显增加，以及平滑肌完整性降低。此外，与各自对照组相比，在第7天（增加约两倍）和第14天（增加约2.5倍）弹性蛋白酶处理的小鼠中，c - FLFLF - Cy7成像探针的荧光强度也显著增加。SPECT成像显示，与对照组相比第14天AAA小鼠中Tc - cFLFLF放射性标记探针的信号强度增加了数倍。用c - FLFLF - Cy5对主动脉组织进行免疫染色显示，与对照组相比，AAA中与中性粒细胞的共表达明显增加。
cFLFLF，一种新型FPR1配体，能够对AAA进行可量化的非侵入性诊断和病情进展评估。使用SPECT成像的这种炎症细胞特异性分子探针的临床应用可能允许AAA形成的早期诊断，从而实现靶向治疗干预并预防即将发生的主动脉破裂。
