Abstract/Description
Introduction: Blood flow restoration to ischemic cardiac tissue is a critical and necessary intervention that has an associated downside: reperfusion injury. Reperfusion injury is a complicated process mediated in part by a pathological increase in reactive oxygen species (ROS). Although extremely elevated levels of ROS can cause cell damage, a minimum level of ROS is necessary for activation of Hydroxylated-HIF-1a (HIF-1a) and inhibition of Peroxisome Proliferator-Activated Receptor-alpha (PPARa) during myocardial ischemic reperfusion injury (MIRI). MIRI clinical manifestations include arrhythmias, myocardial stunning, decreased ventricular contractility and no-reflow. Platelet rich plasma (PRP) has improved left ventricular (LV) function after MIRI in a number of animal models and could be a valuable therapeutic agent against ischemia caused by cardiac and other vascular surgeries. We examined the effect of PRP on LV mechanical function in the Langendorff rabbit heart MIRI model in conjunction with the inhibition of NADPH oxidase NOX-2 or NOX-4. In vitro ROS production studies were performed in cultured H9c2,as well as the interaction of co-cultured adipose derived stem cells (ADSC) and H9c2 cells. Materials: Human PRP was used in all studies. Langendorff Heart Experiments: All spontaneously beating male New Zealand White rabbit hearts were exposed to 30 min of global ischemia and 1 hr. of reperfusion and LV systolic (LVS) and diastolic pressures (LVD) and work function (WF) (LVS x HR) were measured. In vitro Experiments: Co-cultured H9c2 and ADSC cells were assessed for the movement of the 2.5 µM cytoplasmic gap junction permeant dye calcein orange/red or 5 µM of the gap junction impermeant Cell tracker CMFDA from H9c2 cells into ADSC. Hydroxylated-HIF-1α was measured in cultured hypoxic or non-hypoxic H9c2 cells. Fluorometry was used to quantify ROS in H9c2 cells exposed to either 30 min of hypoxia or normoxia. Results: Langendorff Heart Experiments: In hearts treated with PRP alone LVS, LVD and WF did not change significantly. There was no difference in LVS when a NOX-2 inhibitor and PRP were introduced prior to I/R, but LVS increased after 10 and 20 min of reperfusion. LVD and WF were unchanged. NOX-4 inhibition with PRP caused a sustained increase in LVS and decrease in LVD. WF significantly increased above baseline values when the NOX- inhibitor was given but fell significantly during reperfusion. In vitro Experiments:Calcein moved from H9c2 cells into ADSC in a dose dependent manner in the cells treated with PRP, suggesting that PRP can enhance the formation of channels/gap junctions. ROS levels in H9c2 cells exposed to 30 min of hypoxia decreased significantly from the control value in the hypoxic cells but not in a dose dependent manner, with no changes in ROS in normoxic cells. HIF-1α activity was greater in hypoxic H9c2 cells than normoxic cells, suggesting that PRP may help to stabilize HIF-1α, supporting energy production and cell survival. Conclusion: Collectively, these data suggest that PRP may be a therapeutic tool that can mediate ROS production under ischemic/hypoxic conditions. PRP may also support LV mechanical and electrical function by forming gap junctions or channels.
College/School/Affiliation
College of Sciences
Included in
NADPH Oxidase Isoforms (NOX-2 and NOX-4) in Myocardial Ischemia/Reperfusion Injury: Platelet Rich Plasma (PRP) as a Therapeutic Agent
Introduction: Blood flow restoration to ischemic cardiac tissue is a critical and necessary intervention that has an associated downside: reperfusion injury. Reperfusion injury is a complicated process mediated in part by a pathological increase in reactive oxygen species (ROS). Although extremely elevated levels of ROS can cause cell damage, a minimum level of ROS is necessary for activation of Hydroxylated-HIF-1a (HIF-1a) and inhibition of Peroxisome Proliferator-Activated Receptor-alpha (PPARa) during myocardial ischemic reperfusion injury (MIRI). MIRI clinical manifestations include arrhythmias, myocardial stunning, decreased ventricular contractility and no-reflow. Platelet rich plasma (PRP) has improved left ventricular (LV) function after MIRI in a number of animal models and could be a valuable therapeutic agent against ischemia caused by cardiac and other vascular surgeries. We examined the effect of PRP on LV mechanical function in the Langendorff rabbit heart MIRI model in conjunction with the inhibition of NADPH oxidase NOX-2 or NOX-4. In vitro ROS production studies were performed in cultured H9c2,as well as the interaction of co-cultured adipose derived stem cells (ADSC) and H9c2 cells. Materials: Human PRP was used in all studies. Langendorff Heart Experiments: All spontaneously beating male New Zealand White rabbit hearts were exposed to 30 min of global ischemia and 1 hr. of reperfusion and LV systolic (LVS) and diastolic pressures (LVD) and work function (WF) (LVS x HR) were measured. In vitro Experiments: Co-cultured H9c2 and ADSC cells were assessed for the movement of the 2.5 µM cytoplasmic gap junction permeant dye calcein orange/red or 5 µM of the gap junction impermeant Cell tracker CMFDA from H9c2 cells into ADSC. Hydroxylated-HIF-1α was measured in cultured hypoxic or non-hypoxic H9c2 cells. Fluorometry was used to quantify ROS in H9c2 cells exposed to either 30 min of hypoxia or normoxia. Results: Langendorff Heart Experiments: In hearts treated with PRP alone LVS, LVD and WF did not change significantly. There was no difference in LVS when a NOX-2 inhibitor and PRP were introduced prior to I/R, but LVS increased after 10 and 20 min of reperfusion. LVD and WF were unchanged. NOX-4 inhibition with PRP caused a sustained increase in LVS and decrease in LVD. WF significantly increased above baseline values when the NOX- inhibitor was given but fell significantly during reperfusion. In vitro Experiments:Calcein moved from H9c2 cells into ADSC in a dose dependent manner in the cells treated with PRP, suggesting that PRP can enhance the formation of channels/gap junctions. ROS levels in H9c2 cells exposed to 30 min of hypoxia decreased significantly from the control value in the hypoxic cells but not in a dose dependent manner, with no changes in ROS in normoxic cells. HIF-1α activity was greater in hypoxic H9c2 cells than normoxic cells, suggesting that PRP may help to stabilize HIF-1α, supporting energy production and cell survival. Conclusion: Collectively, these data suggest that PRP may be a therapeutic tool that can mediate ROS production under ischemic/hypoxic conditions. PRP may also support LV mechanical and electrical function by forming gap junctions or channels.