Detection of intrathrombotic endothelial progenitor cells and its application to thrombus age estimation in a murine deep vein thrombosis model

Endothelial progenitor cells (EPCs), a newly identified cell type, are bone marrow-derived progenitor cells that co-express stem cell markers and Flk-1 (one of the receptors for vascular endothelial growth factor). In this study, double-color immunofluorescence analysis was performed using anti-CD34 and anti-Flk-1 antibodies in order to examine the time-dependent intrathrombotic appearance of EPCs, using the thrombi of DVT model mice with different thrombus ages (1-21 days). In thrombus cross-section specimens with an age of less than 3 days, CD34+/Flk-1+ EPCs were not detected. EPCs were initially observed in wounds aged 5 days, and their number was increased in thrombi with the advance of thrombus ages. The number of EPCs was the largest in the 10-day thrombus. Moreover, all 15 samples aged 7-14 days had an EPC number of more than 10, and, in 9 of them, the number of intrathrombotic EPCs was over 20. In contrast, in all thrombus samples aged 21 days, the number of intrathrombotic EPCs was less than 20. However, in three of them, the intrathrombotic EPC number was >= 10. These observations suggested that an intrathrombotic EPC number exceeding 20 would indicate a thrombus age of approximately 7-14 days.