Acute preconditioning of cardiac progenitor cells with hydrogen peroxide enhances angiogenic pathways following ischemia-reperfusion injury.

There are a limited number of therapies available to prevent heart failure following myocardial infarction. One novel therapy that is currently being pursued is the implantation of cardiac progenitor cells (CPCs); however, their responses to oxidative stress during differentiation have yet to be elucidated. The objective of this study was to determine the effect of hydrogen peroxide (H2O2) treatment on CPC differentiation in vitro, as well as the effect of H2O2 preconditioning before implantation following ischemia-reperfusion (I/R) injury. CPCs were isolated and cloned from adult rat hearts, and then cultured in the absence or presence of H2O2 for 2 or 5 days. CPC survival was assessed with Annexin V, and cellular differentiation was evaluated through mRNA expression for cardiogenic genes. We found that 100 µM H2O2 decreased serum withdrawal-induced apoptosis by at least 45% following both 2 and 5 days of treatment. Moreover, 100 µM H2O2 treatment for 2 days significantly increased endothelial and smooth muscle markers compared to time-matched untreated CPCs. However, continued H2O2 treatment significantly decreased these markers. Left ventricular cardiac function was assessed 28 days after I/R and I/R with the implantation of Luciferase/GFP(+) CPCs, which were preconditioned with 100 µM H2O2 for 2 days. Hearts implanted with Luciferase/GFP(+) CPCs had significant improvement in both positive and negative dP/dT over I/R. Furthermore, cardiac fibrosis was significantly decreased in the preconditioned cells versus both I/R alone and I/R with control cells. We also observed a significant increase in endothelial cell density in the preconditioned CPC hearts compared to untreated CPC hearts, which also coincided with a higher density of Luciferase(+) vessels. These findings suggest that preconditioning of CPCs with H2O2 for 2 days stimulates neoangiogenesis in the peri-infarct area following I/R injury and could be a viable therapeutic option to prevent heart failure.

Characterization of superoxide dismutases in cardiac progenitor cells demonstrates a critical role for manganese superoxide dismutase.

Transplantation of cardiac progenitor cells (CPCs) is currently in early clinical testing as a potential therapeutic strategy. Superoxide is increased in the ischemic myocardium and poor survival of cells is one of the major limitations of cell transplantation therapy. Superoxide dismutase (SOD) levels were analyzed in c-kit-positive CPCs isolated from rat myocardium to identify their roles in protection against oxidative stress-induced apoptosis in vitro. CPCs were subjected to oxidative stress using xanthine/xanthine oxidase (XXO) and little apoptosis was detected. CPCs contained significantly higher levels of SOD1 and SOD2 as compared with adult cardiac cell types, both at the protein and activity levels. Both SOD1 and SOD2 were increased by XXO at the mRNA and protein level, suggesting compensatory adaptation. Only knockdown of SOD2 and not SOD1 with siRNA sensitized the cells to XXO-apoptosis, despite only accounting for 10% of total SOD levels. Finally, we found XXO activated Akt within 10 min, and this regulated both SOD2 gene expression and protection against apoptosis. Rat CPCs are resistant to superoxide-induced cell death, primarily through higher levels of SOD2 compared to adult cardiac-derived cells. Exposure to superoxide increases expression of SOD2 in an Akt-dependent manner and regulates CPC survival during oxidative stress.

The delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury.

Oxidative stress is increased in the myocardium following infarction and plays a significant role in death of cardiac myocytes, leading to cardiac dysfunction. Levels of the endogenous antioxidant Cu/Zn-superoxide dismutase (SOD1) decrease following myocardial infarction. While SOD1 gene therapy studies show promise, trials with SOD1 protein have had little success due to poor pharmacokinetics and thus new delivery vehicles are needed. In this work, polyketal particles, a recently developed delivery vehicle, were used to make SOD1-encapsulated-microparticles (PKSOD). Our studies with cultured macrophages demonstrated that PKSOD treatment scavenges both intracellular and extracellular superoxide, suggesting efficient delivery of SOD1 protein to the inside of cells. In a rat model of ischemia/reperfusion (IR) injury, injection of PKSOD, and not free SOD1 or empty particles was able to scavenge IR-induced excess superoxide 3 days following infarction. In addition, only PKSOD treatment significantly reduced myocyte apoptosis. Further, PKSOD treatment was able to improve cardiac function as measured by acute changes in fractional shortening from baseline echocardiography, suggesting that sustained delivery of SOD1 is critical during the early phase of cardiac repair. These data demonstrate that delivery of SOD1 with polyketals is superior to free SOD1 protein therapy and may have potential clinical implications.

Sustained release of a p38 inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction.

Cardiac dysfunction following acute myocardial infarction is a major cause of death in the world and there is a compelling need for new therapeutic strategies. In this report we demonstrate that a direct cardiac injection of drug-loaded microparticles, formulated from the polymer poly(cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK), improves cardiac function following myocardial infarction. Drug-delivery vehicles have great potential to improve the treatment of cardiac dysfunction by sustaining high concentrations of therapeutics within the damaged myocardium. PCADK is unique among currently used polymers in drug delivery in that its hydrolysis generates neutral degradation products. We show here that PCADK causes minimal tissue inflammatory response, thus enabling PCADK for the treatment of inflammatory diseases, such as cardiac dysfunction. PCADK holds great promise for treating myocardial infarction and other inflammatory diseases given its neutral, biocompatible degradation products and its ability to deliver a wide range of therapeutics.