Background—Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin 4 (T4) as a mediator of eEPC-mediated cardioprotection.

Methods and Results—In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without T4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5x10⁶ cells) or cells transfected with T4 shRNA when indicated or 15 mg T4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and methylene blue exclusion), and inflammatory cell influx (myeloperoxidase activity) were determined 24 hours later. Survival of neonatal rat cardiomyocytes increased from 32±4% to 90±2% after eEPC application, an effect sensitive to shRNA transfection compared with T4 (45±7%). In vivo, infarct size decreased with eEPC application (38±4% versus 54±4% of area at risk; P<0.01), an effect abolished by T4 shRNA (62±3%). Segmental subendocardial shortening improved after eEPC treatment (22±3% versus -3±4% of control area) unless T4 shRNA was transfected (-6±4%). Retroinfusion of T4 mimicked eEPC application (infarct size, 37±3%; segmental endocardial shortening, 34±7%). Myeloperoxidase activity (3323±388 U/mg in controls) was decreased by eEPCs (1996±546 U/mg) or T4 alone (1455±197 U/mg) but not T4 shRNA–treated eEPCs (5449±829 U/mg).

Conclusion—Our findings show that short-term cardioprotection derived by regional application of eEPCs can be attributed, at least in part, to T4.