Pretreatment of therapeutic cells with poly(ADP-ribose) polymerase inhibitor enhances their efficacy in an in vitro model of cell-based therapy in myocardial infarct.

Abstract

The potential of cell-based therapies in diseases involving ischemia-reperfusion is greatly hampered by the excessive loss of administered cells in the harsh and oxidative environment where these cells are supposed to act. Therefore, we investigated if inhibition of poly(ADP-ribose) polymerase (PARP) in the therapeutically added cells would lead to their increased viability and, subsequently, to an enhanced effect in an in vitro simulated ischemia-reperfusion (I-R) setting. Ischemic conditions were simulated by oxygen and glucose deprivation for 160 min using H9c2 rat cardiomyoblast cells. After 30 min of reperfusion, these cells received 4 types of treatments: no added cells (I-R model), fluorescently labeled (Vybrant DiD) therapeutic H9c2 cells with vehicle (H9c2) or PARP inhibitor (10 microM or 100 microM PJ34) pretreatment. We assessed viability (live, apoptotic and necrotic) of both 'postischemic' and therapeutic cells with flow cytometric analysis using calcein-AM/ethidium homodimer-2 fluorescent staining after 24 h of co-culture. Further measurements on necrosis and metabolic activity were performed using lactate dehydrogenase (LDH) release and resazurin based assays. The percentage of surviving therapeutic cells increased significantly with PARP inhibition (untreated, 52.02+/-5.01%; 10 microM PJ34, 63.38+/-4.50%; 100 microM PJ34, 64.99+/-3.47%). The percentage of necrotic cells decreased in a similar manner (untreated, 37.23+/-4.40%; 10 microM PJ34, 26.83+/-3.49%; 100 microM PJ34, 24.96+/-2.43%). Notably, the survival of the cells that suffered I-R injury was also significantly higher when treated with PARP-inhibited therapeutic cells (I-R model, 36.44+/-5.05%; H9c2, 42.81+/-5.11%; 10 microM PJ34, 52.07+/-5.80%; 100 microM PJ34, 54.95+/-5.55%), while necrosis was inhibited (I-R model, 43.64+/-4.00%; H9c2, 37.29+/-4.55%; 10 microM PJ34, 30.18+/-4.60%; 100 microM PJ34, 25.52+/-3.47%). In subsequent experiments, PARP inhibition decreased LDH-release of the observed combined cell population and enhanced the metabolic activity. Thus, our results suggest that pretreating the therapeutically added cells with a PARP inhibitor could be beneficial in the setting of cell-based therapies.