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 μM or 100 μM 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 μM PJ34, 63.38±4.50%; 100 μM PJ34, 64.99±3.47%). The percentage of necrotic cells decreased in a similar manner (untreated, 37.23±4.40%; 10 μM PJ34, 26.83±3.49%; 100 μM 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 μM PJ34, 52.07±5.80%; 100 μM PJ34, 54.95±5.55%), while necrosis was inhibited (I-R model, 43.64±4.00%; H9c2, 37.29±4.55%; 10 μM PJ34, 30.18±4.60%; 100 μM 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.

Another “String to the Bow” of PJ34, a Potent Poly(ADP-Ribose)Polymerase Inhibitor: An Antiplatelet Effect through P2Y12 Antagonism?

“…So, for example, pre-treatment with 30–1000 nM PJ34 significantly protected neurons from cell death induced by oxygen and glucose deprivation in a dose-dependent manner [21] . PJ34 (10 or 100 µM) also protected cardiomyoblast cells against H 2 O 2 -induced injury [39] . In human umbilical vein endothelial cell culture, Mathews and Berk [40] showed that PJ34 at 10 µM reduced H 2 O 2 -induced cell death.…”

Comparison of the Direct Effects of Human Adipose- and Bone-Marrow-Derived Stem Cells on Postischemic Cardiomyoblasts in anIn VitroSimulated Ischemia-Reperfusion Model

“…For this reason, we have set the length of simulated ischemia to a level where only 5–20% of cells survived without any treatment, which reflects the conditions found at the site of the injury in the heart after myocardial infarction. We demonstrated the suitability of this model by detecting significantly elevated levels of oxidative stress and cellular necrosis after the simulation of ischemia-reperfusion in our earlier publication [42]. The effects were analyzed at 24 hours because we wanted to rule out the potential differentiation of the added stem cells, which occurs over longer time periods.…”

“…Ischemia-reperfusion was simulated in vitro by oxygen and glucose deprivation as described previously in our earlier publications [40–42]. Briefly, 30.000/well H9c2 cells in 12-well plates were incubated in glucose-free DMEM in an atmosphere of 0.5% O 2 and 99.5% N 2 for 160 minutes.…”

Inhibition of GSDMD Activates Poly(ADP-ribosyl)ation and Promotes Myocardial Ischemia-Reperfusion Injury

“…However, persistent excessive activation of PARylation promotes depletion of NAD + and ATP, leading to further cell death [31]. PJ34 is a highly effective PARP inhibitor, and its inhibition of PARP-1 activity is mainly through the competitive blocking of NAD + binding sites on the PARP-1 molecule [32,33].…”