eNOS gene therapy in ischemia -reperfusion injury

Abstract

The protective effect of endothelial nitric oxide synthase (eNOS) derived nitric oxide (NO) in limiting ischemia-reperfusion (I-R) injury has been a central focus of study for a number of years. NO was the first endogenously produced gaseous messenger to be identified and is now well-established throughout the literature as a complex signaling molecule known to possess a number of physiological actions. The purpose of the current studies was to investigate eNOS gene therapy in two clinically relevant models of I-R injury (hepatic and myocardial) and elucidate molecular mechanisms of cellular protection. In the first study we utilized two murine models of transgenic overexpression, a cardiomyocyte-specific eNOS overexpresser (CS-eNOS-Tg) under the control of the alpha-myosin heavy chain promoter and a systemic eNOS transgenic mouse (SYS-eNOS-Tg) under control of the native eNOS promoter. Mice were subjected to in vivo LCA ischemia and reperfusion. CS eNOS-Tg mice displayed significantly decreased infarct size beyond that of mice with systemic overexpression. Additionally, CS eNOS-Tg mice exhibited better preservation of cardiac function as compared to SYS eNOS-Tg mice following myocardial infarction. These results provide valuable evidence that site-specific targeting of eNOS gene therapy may be more advantageous in limiting myocardial I-R injury and subsequent cardiac dysfunction. In the second study, we hypothesized that eNOS gene therapy would augment NO bioavailability and protect against hepatic I-R injury in the setting of type-2 diabetes mellitus. The overexpression of eNOS in the db/db mouse resulted in a profound increase in hepatic injury. Additional experiments suggested that the eNOS enzyme was uncoupled, increasing superoxide production and subsequent hepatocellular injury. Conversely, treatment of diabetic mice with the eNOS cofactor tetrahydrobiopterin (BH4) resulted in a significant decrease in hepatic I-R injury. In this study we present a clear example of both the protective and injurious nature of NO therapy in a well-established model of hepatic I/R injury. We show that, in the setting of diabetes, increasing eNOS via genetic overexpression exacerbates hepatic I/R injury. Our experimental data suggest that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of eNOS enzyme with cofactor therapy may be beneficial.