Haskett Darren G, Maestas David, Howerton Stephen J, Smith Tyler, Ardila D Catalina, Doetschman Tom, Utzinger Urs, McGrath Dominic, McIntyre J Oliver, Vande Geest Jonathan P
1Graduate Interdisciplinary Program of Biomedical Engineering,The University of Arizona,Tucson,AZ 85721,USA.
2Department of Biomedical Engineering,The University of Arizona,Tucson,AZ 85721,USA.
Microsc Microanal. 2016 Apr;22(2):349-60. doi: 10.1017/S1431927616000088. Epub 2016 Feb 23.
Abdominal aortic aneurysm is a multifactorial disease that is a leading cause of death in developed countries. Matrix-metalloproteases (MMPs) are part of the disease process, however, assessing their role in disease initiation and progression has been difficult and animal models have become essential. Combining Förster resonance energy transfer (FRET) proteolytic beacons activated in the presence of MMPs with 2-photon microscopy allows for a novel method of evaluating MMP activity within the extracellular matrix (ECM). Single and 2-photon spectra for proteolytic beacons were determined in vitro. Ex vivo experiments using the apolipoprotein E knockout angiotensin II-infused mouse model of aneurysm imaged ECM architecture simultaneously with the MMP-activated FRET beacons. 2-photon spectra of the two-color proteolytic beacons showed peaks for the individual fluorophores that enable imaging of MMP activity through proteolytic cleavage. Ex vivo imaging of the beacons within the ECM revealed both microstructure and MMP activity. 2-photon imaging of the beacons in aneurysmal tissue showed an increase in proteolytic cleavage within the ECM (p<0.001), thus indicating an increase in MMP activity. Our data suggest that FRET-based proteolytic beacons show promise in assessing MMP activity within the ECM and will therefore allow future studies to identify the heterogeneous distribution of simultaneous ECM remodeling and protease activity in aneurysmal disease.
腹主动脉瘤是一种多因素疾病，是发达国家主要的死亡原因之一。基质金属蛋白酶（MMPs）参与了该疾病的进程，然而，评估它们在疾病起始和进展中的作用一直很困难，动物模型已变得至关重要。将在MMPs存在时激活的荧光共振能量转移（FRET）蛋白水解信标与双光子显微镜相结合，可提供一种评估细胞外基质（ECM）内MMP活性的新方法。在体外测定了蛋白水解信标的单光子和双光子光谱。使用载脂蛋白E基因敲除且输注血管紧张素II的动脉瘤小鼠模型进行的离体实验，同时用MMP激活的FRET信标对ECM结构进行成像。双色蛋白水解信标的双光子光谱显示了各个荧光团的峰值，能够通过蛋白水解切割对MMP活性进行成像。ECM内信标的离体成像揭示了微观结构和MMP活性。动脉瘤组织中信标的双光子成像显示ECM内蛋白水解切割增加（p<0.001），因此表明MMP活性增加。我们的数据表明，基于FRET的蛋白水解信标在评估ECM内的MMP活性方面显示出前景，因此将使未来的研究能够确定动脉瘤疾病中ECM重塑和蛋白酶活性同时存在的异质分布。
