Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands.
J Nucl Med. 2010 Feb;51(2):259-67. doi: 10.2967/jnumed.109.068429.
Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia.
Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using (99m)Tc-labeled AA5 and gamma-imaging were produced in rabbits. (99m)Tc-AA5 uptake after brief ischemia was compared with an I-40/R-180 infarct model. Histologic characterization of both myocardial necrosis and apoptosis was performed in ischemia and infarct models. Phosphatidylserine exteriorization was also studied in a mouse model, and the dynamics and kinetics of phosphatidylserine exposure were assessed using unlabeled recombinant AA5 and AA5 labeled with biotin, Oregon Green, or Alexa 568. Appropriate controls were established.
Phosphatidylserine exposure after ischemia in the rabbit heart could be detected by radionuclide imaging with (99m)Tc-AA5. Pathologic characterization of the explanted rabbit hearts did not show apoptosis or necrosis. Homogenization and ultracentrifugation of the ischemic myocardial tissue from rabbit hearts recovered two thirds of the radiolabeled AA5 from the cytoplasmic compartment. Murine experiments demonstrated that the cardiomyocytes expressed phosphatidylserine on their cell surface after an ischemic insult of 5 min. Phosphatidylserine exposure occurred continuously for at least 6 h after solitary ischemic insult. AA5 targeted the exposed phosphatidylserine on cardiomyocytes; AA5 was internalized into cytoplasmic vesicles within 10-30 min. Twenty-four hours after ischemia, cardiomyocytes with internalized AA5 had restored phosphatidylserine asymmetry of the sarcolemma, and no detectable phosphatidylserine remained on the cell surface. The preadministration of a pan-caspase inhibitor, zVAD-fmk, prevented phosphatidylserine exposure after ischemia.
After a single episode of ischemia, cardiomyocytes express phosphatidylserine, which is amenable to targeting by AA5, for at least 6 h. Phosphatidylserine exposure is transient and internalized in cytoplasmic vesicles after AA5 binding, indicating the reversibility of the apoptotic process.
心肌缺血会导致心肌细胞凋亡。由于细胞凋亡的特征是细胞膜上的磷脂酰丝氨酸外翻，并且 annexin-A5（AA5）能够与磷脂酰丝氨酸紧密结合，因此我们假设放射性标记的 AA5 应该能够识别心肌缺血区域。
通过冠状动脉阻塞 10 分钟（I-10）和再灌注 180 分钟（R-180）来产生短暂心肌缺血的模型，以使用（99m）Tc 标记的 AA5 和γ成像检测磷脂酰丝氨酸外翻。比较短暂缺血后（99m）Tc-AA5 的摄取与 I-40/R-180 梗死模型。在缺血和梗死模型中进行心肌坏死和凋亡的组织学特征分析。还在小鼠模型中研究了磷脂酰丝氨酸外翻，并用未标记的重组 AA5 和用生物素、Oregon Green 或 Alexa 568 标记的 AA5 评估了磷脂酰丝氨酸暴露的动力学和动力学。建立了适当的对照。
（99m）Tc-AA5 放射性核素成像可检测兔心脏缺血后的磷脂酰丝氨酸暴露。从兔心脏取出的心脏标本的病理特征分析未显示凋亡或坏死。从兔心肌缺血组织匀浆和超速离心可从细胞质部分回收三分之二的放射性标记 AA5。在小鼠实验中，在缺血 5 分钟后，心肌细胞在其细胞膜上表达磷脂酰丝氨酸。在单独缺血后，至少在 6 小时内，磷脂酰丝氨酸暴露持续发生。AA5 靶向心肌细胞上暴露的磷脂酰丝氨酸；AA5 在 10-30 分钟内被内化到细胞质囊泡中。缺血后 24 小时，内化 AA5 的心肌细胞恢复了肌浆网的磷脂酰丝氨酸不对称性，细胞表面不再检测到可检测的磷脂酰丝氨酸。预先给予泛半胱天冬酶抑制剂 zVAD-fmk 可防止缺血后磷脂酰丝氨酸暴露。
单次缺血后，心肌细胞表达至少 6 小时的磷脂酰丝氨酸，可通过 AA5 靶向。磷脂酰丝氨酸暴露是短暂的，在与 AA5 结合后被内化到细胞质囊泡中，表明凋亡过程是可逆的。
